Spleen Tyrosine Kinase Activation Research Articles

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.

Spleen tyrosine kinase activity regulates epidermal growth factor receptor signaling pathway in ovarian cancer.

BackgroundSpleen tyrosine kinase (SYK) is frequently upregulated in recurrent ovarian carcinomas, for which effective therapy is urgently needed. SYK phosphorylates several substrates, but their translational implications remain unclear. Here, we show that SYK interacts with EGFR and ERBB2, and directly enhances their phosphorylation.MethodsWe used immunohistochemistry and immunoblotting to assess SYK and EGFR phosphorylation in ovarian serous carcinomas. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. To study its role in EGFR signaling, SYK activity was modulated using a small molecule inhibitor, a syngeneic knockout, and an active kinase inducible system. We applied RNA-seq and phosphoproteomic mass spectrometry to investigate the SYK-regulated EGF-induced transcriptome and downstream substrates.FindingsInduced expression of constitutively active SYK130E reduced cellular response to EGFR/ERBB2 inhibitor, lapatinib. Expression of EGFRWT, but not SYK non-phosphorylatable EGFR3F mutant, resulted in paclitaxel resistance, a phenotype characteristic to SYK active ovarian cancers. In tumor xenografts, SYK inhibitor reduces phosphorylation of EGFR substrates. Compared to SYKWT cells, SYKKO cells have an attenuated EGFR/ERBB2-transcriptional activity and responsiveness to EGF-induced transcription. In ovarian cancer tissues, pSYK (Y525/526) levels showed a positive correlation with pEGFR (Y1187). Intense immunoreactivity of pSYK (Y525/526) correlated with poor overall survival in ovarian cancer patients.InterpretationThese findings indicate that SYK activity positively modulates the EGFR pathway, providing a biological foundation for co-targeting SYK and EGFR.FundDepartment of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, NIH/NCI, Ovarian Cancer Research Foundation Alliance, HERA Women's Cancer Foundation and Roseman Foundation. Funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript and eventually in the decision to submit the manuscript.

Spleen tyrosine kinase (SYK) blocks autophagic Tau degradation in vitro and in vivo

Spleen tyrosine kinase (SYK) plays a major role in inflammation and in adaptive immune responses and could therefore contribute to the neuroinflammation observed in various neurodegenerative diseases. Indeed, previously we have reported that SYK also regulates β-amyloid (Aβ) production and hyperphosphorylation of Tau protein involved in these diseases. Moreover, SYK hyperactivation occurs in a subset of activated microglia, in dystrophic neurites surrounding Aβ deposits, and in neurons affected by Tau pathology both in individuals with Alzheimer's disease (AD) and in AD mouse models. SYK activation increases Tau phosphorylation and accumulation, suggesting that SYK could be an attractive target for treating AD. However, the mechanism by which SYK affects Tau pathology is not clear. In this study, using cell biology and biochemical approaches, along with immunoprecipitation and immunoblotting, quantitative RT-PCR, and ELISAs, we found that SYK inhibition increases autophagic Tau degradation without impacting Tau production. Using neuron-like SH-SY5Y cells, we demonstrate that SYK acts upstream of the mammalian target of rapamycin (mTOR) pathway and that pharmacological inhibition or knockdown of SYK decreases mTOR pathway activation and increases autophagic Tau degradation. Interestingly, chronic SYK inhibition in a tauopathy mouse model profoundly reduced Tau accumulation, neuroinflammation, neuronal and synaptic loss, and also reversed defective autophagy. Our results further suggest that the SYK up-regulation observed in the brains of individuals with AD contributes to defective autophagic clearance leading to the accumulation of pathogenic Tau species. These findings further highlight SYK as a therapeutic target for the treatment of tauopathies and other neurodegenerative proteinopathies associated with defective autophagic clearance.

PS1155 A PHASE I/II STUDY OF THE SYK INHIBITOR ENTOSPLETINIB IN COMBINATION WITH OBINUTUZUMAB IN PATIENTS WITH RELAPSED/REFRACTORY CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

Background:B‐cell receptor (BCR) signaling kinases are important targets in therapy of CLL. However, incomplete responses and emerging resistance to targeted agents justifies development of novel combination strategies. Resistance of lymphoid niche‐resident CLL cells to BCR‐signaling inhibition is, in part, fostered by the soluble mediators present in the tumor microenvironment. Among these, B‐cell activating factor (BAFF) is critical in the survival of both healthy and neoplastic B‐cells. We found that BAFF‐mediated activation of spleen tyrosine kinase (SYK) triggered BCR signaling, thereby contributing to apoptosis resistance in CLL cells. The SYK inhibitor entospletinib (ENTO) abrogated BAFF‐mediated BCR signaling and down‐modulated the anti‐apoptotic protein MCL1 in vitro. Moreover, an anti‐CD20 antibody, obinutuzumab (Obin), downregulates BCLX in neoplastic B‐cells.Aims:Armed with this mechanistic knowledge, we designed a Phase I/II investigator‐sponsored trial of ENTO in combination with Obin in patients (pts) with relapsed/refractory (R/R) CLL and non‐Hodgkin lymphoma (NHL).Methods:Eligible pts were aged ≥18 years, had CLL/NHL (Phase I) or CLL (Phase II), relapsed and/or refractory to ≥1 prior therapies (no prior SYK inhibitor), ECOG performance status ≤2 and preserved organ function. The Phase I part of the study followed a standard 3+3 design with two dose levels (DL1: ENTO 200 mg PO BID; DL2 – ENTO 400 mg PO BID). Obin was given IV on days 1, 2, 8, 15 of Cycle 1 and Day 1 of Cycles 2–6 in standard doses. ENTO was given until disease progression. Primary study objectives were toxicity (Phase I) and efficacy (objective response rate (ORR); Phase II).Results:At DL1 of Phase I, six pts were enrolled (4 ‐ CLL; 2 ‐ follicular lymphoma). One pt experienced a dose‐limiting toxicity (DLT: grade 3 asymptomatic LFT abnormalities which failed to resolve within 72 hours) attributed to ENTO. Other grade 3–4 toxicities included 2 grade 3 infusion reactions and one transient grade 4 neutropenia (attributed to Obin). Four pts remain on therapy after a median follow‐up of 15 months. Three pts were enrolled at DL2 without DLTs.In Phase 2, 18 pts with CLL received ENTO 400 mg PO BID (in combination with Obin). One pt was deemed ineligible due to Richter's transformation at study entry. Of the 17 evaluable pts, 71% were men. Median age was 66 years (range 47–76), and 76% were aged >65 years. 94% had ECOG performance status ≤1. Four pts (24%) had a complex karyotype, 2 (12%) had del(17p) and 8 (48%) had del(13q). Median number of prior therapies was 2 (range, 1–6): 47% had received prior fludarabine and 53% bendamustine. 29% received prior ibrutinib.As of February 1, 2019, with median follow‐up of 8 months (range, 6–14 months), 83% of pts (14/17) remain on treatment. Median relative dose intensity of ENTO (ratio of actual to planned cumulative dose during drug exposure period) was 80%. ORR was 76% (95% CI: 50–93%): 12 (70%) pts with partial response, one (6%) pt had unconfirmed CR. Four (24%) pts had improvement in lymphadenopathy. Median duration of response and median progression‐free survival (PFS) were not reached. All pts are alive. Two (12%) pts discontinued treatment (withdrawal of consent; recurrent LFT abnormalities). The most frequently occurring adverse events of all grades were infusion‐related reactions, neutropenia and fatigue (Table).Summary/Conclusion:In summary, a combination of ENTO and Obin was effective and well tolerated in patients with R/R CLL.

SYK inhibition with fostamatinib in Waldenström's macroglobulinemia: In vitro cell based assays.

e19047 Background: Waldenström's macroglobulinemia (WM) is characterized by accumulation of IgM secreting neoplastic B cells in the bone marrow (BM). B-cell antigen receptor (BCR) signaling plays a pivotal role in the regulation of integrin-mediated retention of lymphoma cells in the BM. Signaling through the BCR leads to activation of spleen tyrosine kinase (SYK) and downstream kinases which play a role in lymphoma pathobiology. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib targets BCR-controlled signaling and integrin-mediated adhesion in WM cells, but patients with WM may become refractory to ibrutinib treatment. As SYK signaling is upstream of BTK, we explored the effect of the SYK inhibitor R406 (the active metabolite of fostamatinib) on BCR mediated signaling and cell adhesion in WM. Methods: MWCL-1 cells were treated with R406, CAL101 (PI3 kinase inhibitor) or ibrutinib at concentrations up to 10µM and stimulated with anti-IgM antibodies or PMA. The cells were transferred to plates coated with fibronectin to assess cell adhesion. For the proliferation assays, MWCL-1 cells or primary B cells were pretreated with kinase inhibitors, followed by stimulation of primary B cells with anti-IgM. Results: Anti-IgM or PMA alone stimulated the adhesion of MWCL-1 on fibronectin. The addition of R406 had a comparable effect to ibrutinib on inhibition of IgM mediated cell adhesion (IC50 = 0.32µM), consistent with the effect of both compounds on BCR mediated signaling. Neither compound affected PMA induced cell adhesion, indicating that the effect on IgM mediated cell adhesion was not due to toxicity. Lack of off-target toxicity was further confirmed in the MWCL-1 proliferation assays where the compounds showed less potency than in BCR-mediated assays. Comparable inhibition of adhesion and proliferation was seen with CAL101. Conclusions: Preliminary preclinical studies with R406 show that SYK is involved in BCR mediated signaling and cell adhesion in WM. CAL101 mediated inhibition of adhesion further confirmed the role of SYK, which is upstream of PI3K. R406 was as effective as ibrutinib in blocking WM cell adhesion. Further studies of SYK inhibition with fostamatinib as a potential treatment option for WM are warranted.

A reevaluation of the spleen tyrosine kinase (SYK) activation mechanism

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.

Spleen Tyrosine Kinase-Mediated Autophagy Is Required for Epithelial-Mesenchymal Plasticity and Metastasis in Breast Cancer.

The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here, we compare a reversible model of epithelial-mesenchymal transition (EMT) induced by TGFβ to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGFβ-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal-epithelial transition (MET). Genetic knockout of autophagy-related 7 (ATG7) or pharmacologic inhibition of SYK activity with fostamatinib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, this study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells. SIGNIFICANCE: These findings present inhibition of spleen tyrosine kinase as a therapeutic option to limit breast cancer metastasis by promoting systemic tumor dormancy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/8/1831/F1.large.jpg.See related commentary by Farrington and Narla, p. 1756.

The orphan nuclear receptor NR4A1 promotes FcεRI-stimulated mast cell activation and anaphylaxis by counteracting the inhibitory LKB1/AMPK axis.

Nuclear receptor subfamily 4 group A member 1 (NR4A1), an orphan nuclear receptor, has been implicated in several biological events such as metabolism, apoptosis, and inflammation. Recent studies indicate a potential role for NR4A1 in mast cells, yet its role in allergic responses remains largely unknown. The aim of this study was to clarify the role of NR4A1 in mast cell activation and anaphylaxis. To evaluate the function of NR4A1 in mast cells, the impacts of siRNA knockdown, gene knockout, adenoviral overexpression, and pharmacological inhibition of NR4A1 on FcεRI signaling and effector functions in mouse bone marrow-derived mast cells (BMMCs) invitro and on anaphylactic responses invivo were evaluated. Knockdown or knockout of NR4A1 markedly suppressed degranulation and lipid mediator production by FcεRI-crosslinked BMMCs, while its overexpression augmented these responses. Treatment with a NR4A1 antagonist also blocked mast cell activation to a similar extent as NR4A1 knockdown or knockout. Moreover, mast cell-specific NR4A1-deficient mice displayed dampened anaphylactic responses invivo. Mechanistically, NR4A1 promoted FcεRI signaling by counteracting the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) axis. Following FcεRI crosslinking, NR4A1 bound to the LKB1/AMPK complex and sequestered it in the nucleus, thereby promoting FcεRI downstream signaling pathways. Silencing or knockout of LKB1/AMPK largely abrogated the effect of NR4A1 on mast cell activation. Additionally, NR4A1 facilitated spleen tyrosine kinase activation independently of LKB1/AMPK. Nuclear receptor subfamily 4 group A member 1 positively regulates mast cell activation by antagonizing the LKB1-AMPK-dependent negative regulatory axis. This finding may provide a novel therapeutic strategy for the development of anti-allergic compounds.

Syk/PECAM-1 Signaling Pathway Mediates Low Shear Stress-Induced Inflammatory in Atherosclerosis

Although blood flow shear stress has been demonstrated to alter the physiology of vascular endothelial cells, little is known about the role of Syk in PECAM-1 signaling pathway exposed to low shear stress (LSS). Methods: In vitro, primary human umbilical vein endothelial cells (HUVECs) were stimulated with parallel plate flow chamber system for 12h under normal shear stress (NSS, 15dyne/cm2), LSS (5dyne/cm2) and high shear stress (HSS, 25dyne/cm2), respectively, followed by inflammatory response analysis. In vivo, atherosclerotic animal models of rat and rabbit were created by feeding with high cholesterol diets. The animals were applied with LSS stimulation by constricting left carotid artery with a blunted needle (0.8mm in diameter). Small molecule R406, a well-demonstrated Syk inhibitor, was applied to the atherosclerotic animal models as well as HUVEC cells. Results: LSS influenced carotid atherosclerosis in the LSS model of rabbits; LSS promoted expression of PECAM-1 and Syk in LSS-treated atherosclerotic rat model; LSS influenced the morphology of abdominal aortic endothelial cells in rats; LSS increased the expression of pPECAM-1, Syk and NF-κB in HUVECs, and inhibition of Syk attenuated LSSinduced inflammatory response. Conclusion: Activation of spleen tyrosine kinase (Syk)/platelet endothelial cell adhesion molecule-1(PECAM-1) signaling pathway was accompanied with inflammatory response induced by LSS stimulation both in vitro and in vivo. This work demonstrated the important role Syk/PECAM1/NF-κB signaling pathway plays in LSS-induced inflammatory response. Funding Statement: This work was supported by Pudong New Area Health Bureau of Shanghai (grant no.PWZbr2017-09), and the National Natural Science Foundation of China (grant no.81401428, 81571693 and 81871361). Declaration of Interests: The authors have declared that no competing interest exists. Ethics Approval Statement: Human umbilical vein from normal full-term newborns was obtained with informed consent and approved in accordance with the procedures of the Medical Ethics Committee of Shanghai East Hospital Affiliated to Tongji University (China).

Spleen tyrosine kinase from Nile tilapia (Oreochromis niloticus): Molecular characterization, expression pattern upon bacterial infection and the potential role in BCR signaling and inflammatory response

Spleen tyrosine kinase (SYK), a member of non-receptor tyrosine kinase family, plays an important role in immune responses against pathogen infection, which is capable of activating B cells signaling pathway and regulating inflammatory response. In this study, Nile tilapia (Oreochromis niloticus) ortholog (OnSYK) was identified and characterized at expression pattern against bacterial infection, function in B cells activation pathway and inflammatory response. The cDNA of OnSYK ORF contained 1851 bp of nucleotide sequence encoding polypeptides of 616 amino acids. The deduced OnSYK protein was highly homologous to other species SYK, containing two SH2 domains and a TyrKc domain. Spatial mRNA expression analysis revealed that OnSYK had wide tissue distribution and was highly expressed in the liver. After challenge of Streptococcus agalactiae (S. agalactiae) in vivo, mRNA expression of OnSYK was significantly up-regulated in the head kidney, spleen and liver. The up-regulation of OnSYK transcript was also displayed in the head kidney and spleen leukocytes stimulation with S. agalactiae and LPS in vitro, which was confirmed at protein level in the head kidney leukocytes by FACS analysis. In addition, after induction with mouse anti-OnIgM monoclonal antibody in vitro, the expressions of OnSYK and its downstream molecules (OnLYN, OnBLNK and OnAP-1) were significantly up-regulated in the head kidney leukocytes, and pharmacological inhibition of SYK activity with inhibitor (P505-15) significantly attenuated the expressions of OnLYN, OnBLNK and OnAP-1. Moreover, upon LPS challenge, the expressions of OnSYK, OnTNF-α, OnIL-6 and OnAP-1 were also up-regulated in the head kidney monocytes/macrophages. After treatment with SYK inhibitor (BAY 61–3606), the expressions of OnTNF-α, OnIL-6 and OnAP-1 were inhibited in the LPS-challenged head kidney monocytes/macrophages. Taken together, the results of this study indicated that OnSYK, playing potential roles in BCR signaling and inflammatory response, was likely to get involved in host defense against bacterial infection in Nile tilapia.

7-Ketocholesterol impairs phagocytosis and efferocytosis via dysregulation of phosphatidylinositol 4,5-bisphosphate.

The plasma membrane is inhomogeneously organized containing both highly ordered and disordered nanodomains. 7-Ketocholesterol (7KC), an oxysterol formed from the nonenzymatic oxidation of cholesterol, is a potent disruptor of membrane order. Importantly, 7KC is a component of oxidized low-density lipoprotein and accumulates in macrophage and foam cells found in arterial plaques. Using a murine macrophage cell line, J774, we report that both IgG-mediated and phosphatidylserine-mediated phagocytic pathways are inhibited by the accumulation of 7KC. Examination of the well-studied Fcγ receptor pathway revealed that the cell surface receptor abundance and ligand binding are unaltered while downstream signaling and activation of spleen tyrosine kinase is not affected. However, while the recruitment of phospholipase Cγ1 was unaffected its apparent enzymatic activity was compromised resulting in sustained phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] levels and polymerized actin at the base of the phagocytic cup. Additionally, we found that 7KC prevented the activation of PLCβ downstream of the P2Y6 G-protein coupled receptor and that 7KC impaired PLCγ activity in response to a direct elevation of cytosolic calcium induced by ionomycin. Finally, we demonstrate that 7KC partly attenuates the activity of rapamycin recruitable constitutively active PLCβ3. Together, our results demonstrate that the accumulation of 7KC impairs macrophage function by altering PtdIns(4,5)P2 catabolism and, thus, impairing actin depolymerization required for the completion of phagocytosis.

Fluorescent Tracking of Yeast Division Clarifies the Essential Role of Spleen Tyrosine Kinase in the Intracellular Control of Candida glabrata in Macrophages.

Macrophages play a critical role in the elimination of fungal pathogens. They are sensed via cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live Candida glabrata in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of C. glabrata did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled C. glabrata were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry. C. glabrata remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of C. glabrata with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage–fungal interaction. In conclusion, we demonstrate a new method of tracking division of C. glabrata using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed C. glabrata.

Inhibition of ovarian tumor cell invasiveness by targeting SYK in the tyrosine kinase signaling pathway.

Ovarian cancer cell motility and invasiveness are prerequisites for dissemination, and largely account for cancer mortality. We have identified an actionable kinase, spleen tyrosine kinase (SYK), which is keenly associated with tumor progression in ovarian cancer. Here, we report that active recombinant SYK directly phosphorylates cortactin and cofilin, which are critically involved in assembly and dynamics of actin filament through phosphorylation signaling. Enhancing SYK activity by inducing expression of a constitutively active SYK mutant, SYK130E, increased growth factor-stimulated migration and invasion of ovarian cancer cells, which was abrogated by cortactin knockdown. Similarly, SYK inhibitors significantly decreased invasion of ovarian cancer cells through basement membrane matrix in a real-time transwell assays and in a 3-D tumor spheroid model. SYK inactivation by gene knockout or by small molecule inhibition reduced actin polymerization. Collectively, the results reported here identify a new mechanism by which SYK signaling regulates ovarian cancer cell motility and invasiveness, and pinpoint a target-based strategy to prevent or suppress the advancement of ovarian malignancies.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response

A family of 11 cell surface-associated aspartyl proteases (CgYps1–11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata. However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1–11Δ mutant. Consistently, the mutant contained lower β-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1–11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1β in THP-1 macrophages. Further, Cgyps1–11Δ–induced IL-1β production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1–11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1β production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1–11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1β production in macrophages.

Methanol extract of Guettarda speciosa Linn. inhibits the production of inflammatory mediators through the inactivation of Syk and JNK in macrophages.

Guettardaspeciosa Linn. (G.speciosa, Rubiaceae) has been used as a traditional medicinal plant in Asia for the treatment of various inflammatory conditions, including cough, fever and maternal postpartum infection. However, the mechanisms underlying the anti‑inflammatory action of G.speciosa extracts have remained elusive. In the present study, the anti‑inflammatory effects of the methanol extract of G.speciosa (MGS) were investigated in murine macrophages by measuring the production of inflammatory mediators and the underlying mechanisms of action by performing immunoblotting analysis of proteins that are potentially involved. MGS reduced nitric oxide (NO) production through regulation of the expression of inducible NO synthase (iNOS) in lipopolysaccharide‑activated RAW264.7 cells; however, cyclooxygenase‑2, the enzyme responsible for prostaglandin E2 production, was not affected at the mRNA or protein level. MGS reduced interleukin‑6 (IL‑6) production, but had no effect on tumor necrosis factor (TNF)‑α production. In addition, MGS suppressed the transcription of IL‑6, but not that of IL‑1β and TNF‑α. The effect of MGS on proinflammatory mediators resulted from the inhibition of the activation of spleen tyrosine kinase and c‑Jun N‑terminal kinase. In conclusion, the present study suggested that MGS may be a potential candidate for development as a therapeutic for alleviating inflammation.

Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release

AimsCalcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. Methods and resultsUsing ELISA to measure IL-1β release from VSMCs, we demonstrated that CaP particles stimulated IL-1β release from proliferating and senescent human VSMCs, but with substantially greater IL-1β release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1β release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1β release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1β and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1β in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1β release. ConclusionsCaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1β, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification.

Interaction of DJ-1 with Lyn is essential for IgE-mediated stimulation of human mast cells

DJ-1 is a redox-sensitive protein with multiple roles in cell homeostasis, levels of which are altered in patients with mast cell (MC)-related disorders. However, whether DJ-1 can regulate human MC function is unknown. We sought to investigate the potential role of DJ-1 in the responses of human MCs to antigen stimulation. DJ-1 was silenced in human CD34+-derived MCs and in the LAD2 MC line by using lentiviral short hairpin RNA constructs. Release of β-hexosaminidase, prostaglandin D2, and GM-CSF and changes in reactive oxygen species levels were measured after FcεRI engagement. Enzymatic assays, sucrose density gradient centrifugation, immunoprecipitation, dot and Western blotting, and confocal imaging were performed for signaling, cellular localization, and coassociation studies. DJ-1 knockdown substantially reduced mediator release, as well as Lyn kinase and spleen tyrosine kinase activation and signaling through mechanisms that appeared largely unrelated to DJ-1 antioxidant activity. Following FcεRI activation, nonoxidized rather than oxidized DJ-1 translocated to lipid rafts, where it associated with Lyn, an interaction that appeared critical for maximal Lyn activation and initiation of signaling. Using purified recombinant proteins, we demonstrated that DJ-1 directly bound to Lyn but not to other Src kinases, and this interaction was specific for human but not mouse proteins. In addition, DJ-1 reduced Src homology 2 domain-containing phosphatase 2 phosphatase activity by scavenging reactive oxygen species, thus preventing spleen tyrosine kinase dephosphorylation and perpetuating MC signaling. We demonstrate a novel role for DJ-1 in the early activation of Lyn by FcεRI, which is essential for human MC responses and provides the basis for an alternative target in allergic disease therapy.

High SYK Expression Drives Constitutive Activation of CD21low B Cells.

Human CD21low B cells present with an activated phenotype and accumulate in distinct disorders connected with chronic immune stimulation. Signaling studies had revealed an increased basal phosphorylation of spleen tyrosine kinase (SYK) and phospholipase Cγ2. Additional BCR stimulation of these constitutively active cells, however, led to reduced activation of these signaling molecules and subsequently NF-κB and Ca2+ activation. In this article, we demonstrate that high SYK expression is a common feature of CD21low B cells independent of the underlying disorder, and that this high expression is sufficient to drive constitutive phosphorylation of SYK and its immediate targets Bruton's tyrosine kinase and phospholipase Cγ2. Inhibition of SYK activity eliminated features of the constitutive activation in these cells and partly restored BCR signaling. High SYK expression is especially induced by CpG or CD40L in combination with IL-21, but not BCR stimulation, suggesting the importance of the immune-stimulatory context for the induction of this B cell phenotype. In summary, high SYK expression is a common feature of human CD21low B cells and presumably results from chronic activation in inflammatory environments present in a subgroup of patients with heterogeneous disorders like chronic infection, autoimmunity, and immunodeficiency. High SYK expression by itself drives the constitutive activation observed in these B cells, which in turn may contribute to the hyporesponsiveness upon BCR stimulation. Given the high prevalence of autoreactive clones among CD21low B cells in autoimmune disorders, the dominant role of SYK in CD21low B cells may provide a new option for therapeutic interventions in patients with expanded CD21low B cells and humoral autoimmunity.

Abstract NTOC-115: ASSOCIATION BETWEEN NUCLEAR SYK PHOSPHORYLATION AND EGFR/ERBB2 PATHWAY AND POOR SURVIVAL IN HIGH GRADE SEROUS OVARIAN CANCER

Abstract BACKGROUND: Spleen tyrosine kinase (SYK) has emerged as a cancer associated kinase of which aberrant activity contributes to tumor development and drug resistance in several types of human cancer including ovarian carcinoma. To better understand the mechanism how the SYK pathway participates in tumor progression, we studied the biochemistry and transcriptional regulation of SYK in high-grade ovarian serous carcinoma. METHODS: Immunohistochemical analyses were performed for phosphorylated SYK (Y525/526) and total SYK levels in 42 primary serous ovarian carcinomas and 112 tumor ascites formalin-fixed paraffin embedded tissues. The relative expression of SYK long and short isoforms in peritoneal ascites and cell lines were examined by reverse transcriptase-PCR. Transcriptome analysis of gene regulated by SYK inhibition was carried out using Illumina Human HT-12 v4 arrays. Results from array was analyzed for upstream regulators. IHC data was correlated with pEGFR (Y1197) and pERBB2 (Y877) nuclear expression. Association with survival was determined by Kaplan-Meier analysis and the log-rank test. RESULTS: We observed that active phosphorylated SYK (p-SYK, Y525/526) is mainly localized in the nucleus and cytoskeleton in cell lines and tissues. Investigation of SYK mRNA spliced variants in ascites tumors showed predominant SYK long isoform expression, consistent with its nuclear localization. Survival analyses revealed that intense nuclear p-SYK correlated with poor overall survival in two ovarian carcinoma cohorts. Patients with relatively high p-SYK in the nucleus had approximately half the median survival compared to those with undetectable or low nuclear p-SYK. SYK inhibition affected changes in gene expression that are downstream of ERBB2 and EGFR signaling. Correlation analyses in tissues suggested a relationship between p-SYK and pERBB2 or pEGFR signaling. CONCLUSIONS: Our results suggest that activation of the SYK pathway leads to its phosphorylation and localization in cell nucleus. SYK activity plays a role in regulating EGFR and ERBB2 phosphorylation and activation. Thus, SYK may regulate tumor-promoting gene transcription through cross-talks with the EGFR/ERBB2 pathways. Citation Format: Yu, Yu; Ardighieri, Laura; Suryo Rahmanto, Yohan; Chen, Lin Y.; Tessarollo, Nayara G.; Mizushima, Taichi; Ayhan, Ayse; Davidson, Ben; Wang, Tian L., and Shih Ie M. ASSOCIATION BETWEEN NUCLEAR SYK PHOSPHORYLATION AND EGFR/ERBB2 PATHWAY AND POOR SURVIVAL IN HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-115.