Spleen Tyrosine Kinase Activation Research Articles

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

Elucidating the Role of SYK in the Intestinal Epithelium

Background: Inflammatory Bowel Disease (IBD) involves chronic and recurring inflammation of the gastrointestinal tract. We identified several patients presenting with immune dysregulation and systemic inflammation, including inflammation of the gut. Analysis of Whole Exome Sequencing data from pediatric IBD patients has identified five rare protein coding Spleen Tyrosine Kinase (SYK) variants. Functional studies have demonstrated increased kinase activity implicating them in disease pathophysiology. While the role of SYK is well characterized in immune cells, it is not understood what role it plays in the intestinal epithelium. Aims: The primary aim of this project is to understand the role SYK plays in the intestinal epithelium, what molecular pathways it signals through, and how its dysregulation leads to intestinal disease. Methods: To understand SYKs role in the intestinal epithelium, a BioID screen was used to identify novel SYK interacting proteins. BioID was done with wild type SYK as well as the patient derived variants in HEK293 cells. BioID hits were validated using co-immunoprecipitation (CoIP) and proximity ligation assay (PLA). Further functional analysis was done in Caco2 cells, an intestinal epithelial cell model, with western blot, ELISA, and luciferase assays to assess the effect of SYK activity on downstream inflammatory pathways. Finally, we investigated the effect of small molecule kinase inhibitors to explore possible novel treatment options. Results: The BioID screen revealed several high confidence interactors, with dysregulated interactions in the patient variants. USP25, a ubiquitin specific peptidase that is widely expressed, including in the intestinal epithelium, was identified as one of the strongest hits based on peptide counts. The interaction was validated with CoIP and PLA, and further analysis showed SYK activation decreased USP25 protein levels by up to eight-fold. The secretion of the proinflammatory chemokine, IL-8, in response to lipopolysaccharide (LPS) was found to be almost twice as high in Caco2 cells with patient SYK variants relative to WT. Cells expressing the patient variants of SYK showed increased NFkB and AP1 levels, indicating increased inflammatory gene expression when stimulated with 100ng/mL LPS. NFkB was also active for longer in cells expressing the patient variant of SYK post stimulation, with WT cells returning to baseline in under half an hour, while the patient variant stayed in an inflamed state for 36 to 48 hours after LPS was removed. Conclusions: Increased SYK activity is associated with the degradation of USP25. USP25 is known to cleave ubiquitin off TNF receptor associated factors (TRAFs). TRAFs are activated downstream of innate immune receptors such as TLR4, and need to be ubiquitinated for activation. USP25, which is responsible for the deubiquitination of these TRAFs, acts as a negative regulator of these pathways. The hyperactive SYK variants degrade USP25 and impair the cells’ ability to switch off inflammatory signaling pathways, indicated by the cells elongated period in an inflamed state. This research is funded by grants from Canadian Institute for Health Research (CIHR) and the Leona M. and Harry B. Helmsley Charitable Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Microglia rely on SYK signalling to mount neuroprotective responses in models of Alzheimer's disease and multiple sclerosis.

Microglia rely on SYK signalling to mount neuroprotective responses in models of Alzheimer's disease and multiple sclerosis.

Disease-specific expansion of CD29+IL-17RA+ T effector cells possessing multiple signalling pathways in spondyloarthritis.

T cells adhere to enthesis fibrocartilage via integrins and intrinsically require IL-17RA-mediated signals to maintain their effector function. We analysed CD29+IL-17RA+ T cells in inflamed lesions and peripheral blood in patients with SpA and investigated their association with disease activity and therapeutic response. Transcriptome analysis of synovial fluid T cells from PsA was performed using publicly available bulk cell RNA sequencing data. Blood samples were obtained from healthy controls (n = 37), RA (n = 12), IgG4-related disease (IgG4-RD; n = 12), large vessel vasculitis (LVV; n = 12) and SpA (n = 28) and were analysed by flow cytometry. T cells in the inflamed joints of PsA showed CD29 and IL-17RA expression. CD29+IL-17RA+ T cells showed enriched CXCR3+CD45RA+ effector cells and activation of spleen tyrosine kinase (Syk), nuclear factor κB (NF-κB) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. The proportion of peripheral blood CD29+IL-17RA+ T cells was significantly increased in patients with SpA compared with patients with RA, IgG4-RD or LVV and in healthy controls. Based on the ASDAS-CRP scores, the proportion of CD29+IL-17RA+ T cells was positively correlated with disease activity in treatment-naïve patients with active SpA. Anti-IL-17 but not anti-TNF monoclonal antibodies reduced CD29+IL-17RA+ T cells. CD29+IL-17RA+ T effector cells with enhanced Syk, NF-κB and JAK-STAT pathways were specifically increased in SpA and were correlated with disease activity, implicating a role of this newly identified T cell population in the pathogenesis. Anti-IL-17 monoclonal antibodies may be effective for patients by reducing this pathogenic T cell population.

Spleen tyrosine kinase mediates innate and adaptive immune crosstalk in SARS‐CoV‐2 mRNA vaccination

Durable cell‐mediated immune responses require efficient innate immune signaling and the release of pro‐inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS‐CoV‐2 mRNA vaccination primes human monocyte‐derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3‐driven pyroptotic cell death and subsequently secrete mature interleukin‐1β. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C‐type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage‐driven activation of effector memory T cells. Furthermore, vaccination‐induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime‐boost concepts to augment innate immune signaling in SARS‐CoV‐2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein‐specific T cell responses.

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.

Polyaniline-Based Glyco-Condensation on Au Nanoparticles Enhances Immunotherapy in Lung Cancer.

Lung cancer is considered among the deadliest cancers with a poor prognosis. Au@PG nanoparticles (NPs) are gold (Au)-based NPs featuring a polyaniline-based glyco structure (PG) generated from the polymerization of ortho-nitrophenyl-β-d-galactopyranoside (ONPG) with promising M1 macrophage polarization activity, resulting in tumor remodeling and from a cold to a hot microenvironment, which promotes the cytotoxic T cell response and tumor inhibition. The combination of Au@PG NPs and anti-programmed cell death protein 1 (PD-1) therapy improved tumor inhibition and immunosuppression, accompanied by the secretion of immunogenic cytokines. A one-pot synthetic method was developed to achieve glyco-condensation during the formation of Au@PG NPs, which induced macrophage polarization more efficiently than Au@glucose, Au@mannose, and Au@galactose NPs. The switch from M2 to M1 macrophages was dependent on NP size, with smaller Au@PG NPs performing better than larger ones, with effectiveness ranked as follows: 32.2 nm ≈ 29.8 nm < 26.4 nm < 18.3 nm. Cellular uptake by endocytosis induced size-dependent endoplasmic reticulum (ER) stress, which resulted in the activation of spleen tyrosine kinase (SYK), leading to immune modulations and macrophage polarization. Our results suggested the promising potential of Au@PG NPs in lung cancer immunotherapy.

Knockdown and Inhibition of Spleen Tyrosine Kinase Decreases mTORC1 Activity and Enhances Lysosomal Biogenesis and Autolysosomal Maturation in Pancreatic Cancer Cell Lines

In cancer, oncogene dependency is a phenomenon where a dominant driver oncogene promotes tumor cell proliferation and survival, and loss of this oncogene results in tumor cell death and, eventually, tumor regression. KRAS, a GTPase that regulates cell growth and proliferation, is an oncogene constitutively activated in over 90% of pancreatic cancer. However, clinically effective inhibitors of KRAS have been unsuccessful so efforts have been focused on identifying other potential targets associated with the KRAS signaling network. Spleen tyrosine kinase (SYK), which is expressed at high levels in KRAS‐dependent pancreatic cancer cell lines, may be one of these targets. Our data indicate that SYK activates the mechanistic target of rapamycin kinase complex 1 (mTORC1), which promotes protein translation and cell growth. SYK activation also leads to decreased autolysosome count. In connecting SYK activation with increased mTORC1 activity and decreased autolysosome count, we hypothesis that the MiT/TFE transcription factors are involved. We propose that SYK inhibition in pancreatic cancer cells leads to reduced mTORC1 activity, which reduces phosphorylation of MiT/TFE transcription factors. The unphosphorylated MiT/TFE transcription factors may then enter the nucleus to activate genes for lysosomal biogenesis and autophagy. Autophagy is a process that recycles cellular macromolecules during nutrient deprivation by fusing autophagosomes and lysosomes, produced from lysosomal biogenesis, to generate autolysosomes. From our experiments, we were able to show that SYK inhibition blocks mTORC1‐dependent phosphorylation of MITF and TFEB transcription factors, members of the MiT/TFE family. MITF and TFEB activation leads to increased autophagy due to autolysosomal biogenesis and accumulation. In summary, our studies of the SYK‐mTORC1‐autophagy pathway provide support to investigate SYK as a candidate therapeutic target for pancreatic cancer treatment.

Amelioration of Lupus Serum-Induced Skin Inflammation in CD64-Deficient Mice.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder characterized by high autoantibodies levels and multiorgan tissue damage. The current study investigated the role of CD64 in SLE patients and animal models. According to a flow cytometry study, SLE patients showed an increase in CD64 expression in circulating monocytes. There was a correlation between CD64 and SLEDAI, blood urea nitrogen levels, and anti-Sm antibodies. In skin lesions of lupus MRL/lpr mice, there was high IgG deposition and CD64 expression. In vitro, cytokines IL-10 and IFN-γ upregulated CD64 expression in monocytes/macrophages that was inhibited by glucocorticoids. In CD64-deficient mice, skin inflammation induced by lupus serum was reduced. Furthermore, activation of spleen tyrosine kinase (Syk), Akt, and extracellular signal-regulated kinase (Erk) was inhibited in CD64-deficient monocytes. The results suggest that CD64 could be a biomarker for observing SLE progression, as well as a mechanistic checkpoint in lupus pathogenesis.

Nutraceutical Aid for Allergies - Strategies for Down-Regulating Mast Cell Degranulation.

Interactions of antigens with the mast cell FcεRI-IgE receptor complex induce degranulation and boost synthesis of pro-inflammatory lipid mediators and cytokines. Activation of spleen tyrosine kinase (Syk) functions as a central hub in this signaling. The tyrosine phosphatase SHP-1 opposes Syk activity; stimulation of NADPH oxidase by FcεRI activation results in the production of oxidants that reversibly inhibit SHP-1, up-regulating the signal from Syk. Activated AMPK can suppress Syk activation by the FcεRI receptor, possibly reflecting its ability to phosphorylate the FcεRI beta subunit. Cyclic GMP, via protein kinase G II, enhances the activity of SHP-1 by phosphorylating its C-terminal region; this may explain its inhibitory impact on mast cell activation. Hydrogen sulfide (H2S) likewise opposes mast cell activation; H2S can boost AMPK activity, up-regulate cGMP production, and trigger Nrf2-mediated induction of Phase 2 enzymes – including heme oxygenase-1, whose generation of bilirubin suppresses NADPH oxidase activity. Phycocyanobilin (PCB), a chemical relative of bilirubin, shares its inhibitory impact on NADPH oxidase, rationalizing reported anti-allergic effects of PCB-rich spirulina ingestion. Phase 2 inducer nutraceuticals can likewise oppose the up-regulatory impact of NADPH oxidase on FcεRI signaling. AMPK can be activated with the nutraceutical berberine. High-dose biotin can boost cGMP levels in mast cells via direct stimulation of soluble guanylate cyclase. Endogenous generation of H2S in mast cells can be promoted by administering N-acetylcysteine and likely by taurine, which increases the expression of H2S-producing enzymes in the vascular system. Mast cell stabilization by benifuuki green tea catechins may reflect the decreased surface expression of FcεRI.

TRIM31 facilitates K27-linked polyubiquitination of SYK to regulate antifungal immunity

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31−/− mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

HECT E3 Ubiquitin Ligase Nedd4 Is Required for Antifungal Innate Immunity.

Candida albicans is the most common cause of fungal infections in humans, and disseminated candidiasis has become one of the leading causes of hospital-acquired bloodstream infections with a high mortality rate. However, little is known about the host-pathogen interactions and the mechanisms of antifungal immunity. Here, we report that Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) is essential for signaling through Dectin-1 and Dectin-2/3. We showed that mice that lack Nedd4 globally or only in the myeloid compartment are highly susceptible to systemic C. albicans infection, which correlates with heightened organ fungal burden, defective inflammatory response, impaired leukocyte recruitment to the kidneys, and defective reactive oxygen species expression by granulocytes. At the molecular level, Nedd4 -/- macrophages displayed impaired activation of TGF-β-activating kinase-1 and NF-κB, but normal activation of spleen tyrosine kinase and protein kinase C-δ on C. albicans yeast and hyphal infections. These data suggest that Nedd4 regulates signaling events downstream of protein kinase C-δ but upstream of or at TGF-β-activating kinase-1.

Nilvadipine suppresses inflammation via inhibition of P-SYK and restores spatial memory deficits in a mouse model of repetitive mild TBI

Repeated exposure to mild TBI (mTBI) has been linked to an increased risk of Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE) and other neurodegenerative diseases. Some pathological features typically observed in AD have been found in postmortem brains of TBI and CTE, hence treatments tested for AD have a potential to be effective against r-mTBI outcomes. Neuroinflammation may present a possible answer due to its central role both in acute brain injury and in chronic degenerative-like disorders. Our previous studies have shown that drug nilvadipine, acting as an inhibitor of spleen tyrosine kinase (SYK), is effective at reducing inflammation, tau hyperphosphorylation and amyloid production in AD mouse models. To demonstrate the effect of nilvadipine in the absence of age-related variables, we introduced the same treatment to young r-mTBI mice. We further investigate therapeutic mechanisms of nilvadipine using its racemic properties. Both enantiomers, (+)-nilvadipine and (−)-nilvadipine, can lower SYK activity, whereas (+)-nilvadipine is also a potent L-type calcium channel blocker (CCB) and shown to be anti-hypertensive. All r-mTBI mice exhibited increased neuroinflammation and impaired cognitive performance and motor functions. Treatment with racemic nilvadipine mitigated the TBI-induced inflammatory response and significantly improved spatial memory, whereas (−)-enantiomer decreased microgliosis and improved spatial memory but failed to reduce the astroglial response to as much as the racemate. These results suggest the therapeutic potential of SYK inhibition that is enhanced when combined with the CCB effect, which indicate a therapeutic advantage of multi-action drugs for r-mTBI.

Research Advance on the Role of Spleen Tyrosine Kinase Inhibitors in Hematologic Malignancies

Abstract Spleen tyrosine kinase (SYK) is not only a key kinase in the B-cell receptor (BCR) signaling pathway, but also a critical component of other signal transduction pathways such as Fc receptor, complement receptor and integrin. Abnormal activation of SYK closely related to the occurrence and development of hematological malignancies, thus targeting SYK has become a research hotspot. Several SYK inhibitors including Fostamatinib, Entospletinib and Cerdulatinib were being evaluated in clincal trials. As a second generation SYK inhibitor, Entospletinib has achieved good efficacy in lymphoid and myeloid hematologic tumors. Furthermore, Entospletinib can significantly relieve hematopoietic stem cell transplantation(HCT) related graft versus host disease (GVHD). In this review the role of SYK inhibitors in treatment of hematological malignancies is summarized brifely.

Synedrella nodiflora (Linn.) Gaertn. inhibits inflammatory responses through the regulation of Syk in RAW 264.7 macrophages.

Synedrella nodiflora (Linn.) Gaertn. (S. nodiflora) has long been used for the treatment of inflammatory diseases, including liver disease, asthma, rheumatism and earache, in tropical countries throughout America, Asia and Africa. However, the biological effects of S. nodiflora have not been extensively studied at the molecular level. Notably, it remains unclear how S. nodiflora exerts anti-inflammatory activity. In the present study, the anti-inflammatory mechanism of a methanol extract of S. nodiflora (MSN) in RAW 264.7 macrophages activated by lipopolysaccharide (LPS) was investigated. Non-cytotoxic concentrations of MSN (≤400 µg/ml) decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which resulted in a decrease in nitric oxide and prostaglandin E2 (PGE2) production. The mRNA expression of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α was reduced upon MSN treatment. In addition, the activation of spleen tyrosine kinase (Syk) and Akt was suppressed by MSN. Taken together, these findings recommend the traditional medicinal application of S. nodiflora in the treatment of several inflammation-associated diseases and indicate the possibility of MSN as a novel therapeutic reagent of inflammation-related diseases.

Neuronal Spleen tyrosine kinase (SYK) mediates cytokine release in Transgenic Tau P301S mice organotypic brain slice cultures

The Spleen Tyrosine Kinase (SYK) is known for its involvement in B-cell and T-cell signaling, modulating the peripheral immune response. We have previously shown that SYK is overactive in the brains of human Alzheimer’s Disease (AD) patients, as well as mouse models of AD and tauopathy including Tg Tau P301S mice. More specifically, SYK activation occurs mainly in neurons in human AD brain specimens and mouse models of AD and colocalizes with tau pathogenic species, suggesting it could play a role in AD pathobiology. To assess the possible contribution of SYK to the inflammatory response induced by tau pathology, we analyzed cytokine production in organotypic brain slices cultures from Tg Tau P301S mice and wild-type littermates. Organotypic brains slices from Tau P301S mice produce more cytokines than brain slices from wild-type littermates while SYK inhibition completely antagonizes cytokine production from Tg Tau P301S brain slices. Interestingly, LPS exacerbates the production of pro-inflammatory cytokines in Tg Tau P301S brain sections compared to wild-type organotypic sections while SYK inhibition alleviates the release of pro-inflammatory cytokines induced by LPS. Given that SYK is mainly activated in neurons in Tg Tau P301S mice and not in glial cells, these data suggest that neuronal SYK contributes to the neuroinflammation triggered by the tau pathology. SYK represents an attractive target for regulating the underlying neuroinflammatory component induced by tau pathology.

Knockdown and Inhibition of SYK Tyrosine Kinase Decreases mTORC1 Activity and Enhances Lysosomal Biogenesis and Autolysosomal Maturation in Pancreatic Cancer Cell Lines

In cancer, oncogene dependency is a phenomenon where a dominant driver oncogene promotes tumor cell proliferation and survival, and loss of this oncogene results in tumor cell death and, eventually, tumor regression. KRAS, a GTPase that regulates cell growth and proliferation, is an oncogene constitutively activated in over 90% of pancreatic cancer. However, clinically effective inhibitors of KRAS have been unsuccessful so efforts have been focused on identifying other potential targets associated with the KRAS signaling network. Spleen tyrosine kinase (SYK), expressed at high levels in KRAS‐dependent pancreatic cancer cell lines, may be one of these targets. Our data indicate that SYK activates the mechanistic target of rapamycin kinase complex 1 (mTORC1), which promotes protein translation and cell growth. SYK activation also leads to decreased autolysosome count. In connecting SYK activation with increase mTORC1 activity and decrease autolysosome count, we hypothesis that the MiT/TFE transcription factors are involved. We propose that SYK inhibition in pancreatic cancer cells leads to reduced mTORC1 activity, which reduces phosphorylation of MiT/TFE transcription factors. The unphosphorylated MiT/TFE transcription factors are then allowed into the nucleus to activate genes for lysosomal biogenesis and autophagy. Autophagy is a process that recycles cellular macromolecules during nutrient deprivation by fusing autophagosomes and lysosomes, produced from lysosomal biogenesis, to generate autolysosomes. From our experiments, we were able to show that SYK inhibition blocks mTORC1‐dependent phosphorylation of MITF and TFEB transcription factors, members of the MiT/TFE family. MITF and TFEB activation leads to increased autophagy due to autolysosomal biogenesis and accumulation. In summary, our studies of the SYK‐mTORC1‐autophagy pathway provide support to investigate SYK as a candidate, therapeutic target for pancreatic cancer treatment.Support or Funding InformationNIH/NCI K99/R00 ‐ Anurag Singh (PI); Boston University Dahod Breast Cancer Pilot Award ‐ Anurag Singh (PI); Boston University's Undergraduate Research Opportunities Program ‐ Michelle Pan

SYK Inhibitor Entospletinib in Combination with Obinutuzumab Demonstrates Efficacy in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

SYK Inhibitor Entospletinib in Combination with Obinutuzumab Demonstrates Efficacy in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

WZ3146 inhibits mast cell Lyn and Fyn to reduce IgE-mediated allergic responses in vitro and in vivo

Mast cells (MCs) play an important role as effector cells that cause allergic responses in allergic diseases. For these reasons, MC is considered an attractive therapeutic target for allergic disease treatment. In this study, we investigated the inhibitory effect of WZ3146, N-[3-[5-chloro-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]prop-2-enamide, and the mechanisms of its actions on the MC activation and IgE-mediated allergic response by using three types of MCs such as rat basophilic leukemia (RBL)-2H3 cells, mouse bone marrow mast cells (BMMCs), and human Laboratory of Allergic Diseases 2 (LAD2) cells. WZ3146 inhibited antigen-stimulated degranulation in a dose-dependent manner (IC50, ~ 0.35 μM for RBL-2H3 cells; ~ 0.39 μM for BMMCs; ~ 0.41 for LAD2 cells). WZ3146 also suppressed the production of histamine, tumor necrosis factor (TNF)-α and interleukin (IL)-6, which mediate various allergic responses, in a dose-dependent manner. As the mechanism of WZ3146 to inhibit MCs, it inhibited the activation of spleen tyrosine kinase (Syk) and the downstream signaling proteins of Syk such as linker for activation of T cell (LAT) and phospholipase (PL) Cγ1 in the signaling pathway of FcεRI. In addition, WZ3146 inhibited the activation of Akt, extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). However, WZ3146 did not inhibit degranulation of MCs by thapsigargin or ionomycin, which increase calcium concentration in cytosol. Notably, WZ3146 inhibited the activity of Lyn and Fyn, but not Syk. In an following animal experiment, WZ3146 inhibited IgE-dependent passive cutaneous anaphylaxis (PCA) in a dose-dependent manner (ED50, ~ 20 mg/kg). Taken together, in this study we show that the pyrimidine derivative, WZ3146, inhibits the IgE-mediated allergic response by inhibiting Lyn and Fyn Src-family kinases, which are initially activated by antigen stimulation in MCs. Therefore, we propose that WZ3146 could be used as a new therapeutic agent for the treatment of allergic diseases.