Статті в журналах: "Syk Inhibitor"

1

Zhao, Xiaoxian, Andrew E. Schade, and Eric Hsi. "Distinct Role of Src Family Kinase Inhibitors in Burkitt Lymphoma Cells Vs. Diffuse Large B-Cell Lymphoma Cells." Blood 112, no. 11 (November 16, 2008): 3765. http://dx.doi.org/10.1182/blood.v112.11.3765.3765.

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Abstract Introduction: The Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of malignancies, with approximately 85% of NHL belonging to the B-cell lineage. Src family kinases (SFKs) are non-receptor intracellular tyrosine kinases which are important in the regulation of multiple signaling pathways including cell proliferation, tumor invasiveness, angiogenesis and apoptosis. Syk is another predominant tyrosine kinase expressed in B-cell lines in addition to SFKs. We attempted to correlate SFK and Syk inhibitor efficacy with the presence of phospho-SFK or phospho-Syk in lymphoma cell lines and tissues. Methods: Cell proliferation was measured with WST-1 reagent. Apoptotic assay was performed with Annexin-V and 7-AAD by flow cytometry (FC, FACSCalibur, BD Bioscience). Phospho-Src (Y416) antibody (cell signaling Technology, CSL) was used for immunoblotting and immunohistochemistry. (IHC, Discovery, Ventana Medical Systems). Phospho-Syk (Y525/526) antibody (CSL) was used for FC and immunoblotting. Results: In a screening for the effects of different kinases’ inhibitors on B-cell lymphoma lines, we observed that SFK inhibitors, PP2 and dasatinib (Sprycel, Bristol Myers Squibb), inhibited proliferation and caused dose-dependent apoptosis induction at 24 h (PP2: 31% at 10 mM; dasatinib: 39% at 100 nM) in Burkitt’s lymphoma cell line Raji. The apoptotic induction was associated with cleavage of caspase-3 and caspase-8. The ability of SFK inhibitors to induce apoptosis in Raji cells paralleled high level expression of constitutive phospho- SFK (Y416). In contrast to this Burkitt’s line, diffuse large B-cell lymphoma (DLBCL) lines (Sud-HL4, Sud-HL-6 and OCI-LY3, OCI-LY10) were less-sensitive to these SFK inhibitors but showed apoptosis induction upon exposure to the Syk inhibitor (piceatannol & syk inhibitor IV). Interestingly, the DLBCL lines that were resistant to SFK inhibitors had undectable or low levels of phospho-SFK (Y416); while their susceptibility to the Syk inhibitor-induced apoptosis paralleled detectable constitutive phospho-Syk (Y525/526). Immunohistochemical staining of burkitt’s lymphoma tissues and a tissue microarray panel of NHL indicated 13/20 (65%) of Burkitt’s lymphoma, 3/5 of small lymphocytic lymphoma, 2/5 of mantle cell lymphoma, 3/10 of follicular lymphoma, 2/5 of DLBCL, 2/5 of marginal zone lymphoma, 1/5 of lymphoblastic lymphoma are positive for phospho-Src (Y416). Staining of normal tonsil tissue showed germinal center cells are strong positive for phospho-Src (Y416), while marginal zone cells are weak positive and plasma cells are negative. We are currently testing the correlation of phospho-Src (Y416) expression in fresh NHL tissues and their sensitivity to Src family kinase inhibitors. Conclusion: These data suggest that rational application of molecularly targeted therapy for aggressive NHL is possible by directly examining key signaling nodes promoting survival and proliferation. For instance, the clinical SFK inhibitor dasatinib is currently being examined in a clinical trial for NHL (NCT00550615). Our results suggest that profiling patients’ lymphoma cells for phospho-SFK could optimize therapeutic efficacy and minimize unnecessary treatment-related side effects.

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Makhoul, Stephanie, Stephanie Dorschel, Stepan Gambaryan, Ulrich Walter, and Kerstin Jurk. "Feedback Regulation of Syk by Protein Kinase C in Human Platelets." International Journal of Molecular Sciences 21, no. 1 (December 25, 2019): 176. http://dx.doi.org/10.3390/ijms21010176.

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The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.

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3

Coates, Matthew S., Eric W. F. W. Alton, Garth W. Rapeport, Jane C. Davies, and Kazuhiro Ito. "Pseudomonas aeruginosa induces p38MAP kinase-dependent IL-6 and CXCL8 release from bronchial epithelial cells via a Syk kinase pathway." PLOS ONE 16, no. 2 (February 1, 2021): e0246050. http://dx.doi.org/10.1371/journal.pone.0246050.

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Pseudomonas aeruginosa (Pa) infection is a major cause of airway inflammation in immunocompromised and cystic fibrosis (CF) patients. Mitogen-activated protein (MAP) and tyrosine kinases are integral to inflammatory responses and are therefore potential targets for novel anti-inflammatory therapies. We have determined the involvement of specific kinases in Pa-induced inflammation. The effects of kinase inhibitors against p38MAPK, MEK 1/2, JNK 1/2, Syk or c-Src, a combination of a p38MAPK with Syk inhibitor, or a novel narrow spectrum kinase inhibitor (NSKI), were evaluated against the release of the proinflammatory cytokine/chemokine, IL-6 and CXCL8 from BEAS-2B and CFBE41o- epithelial cells by Pa. Effects of a Syk inhibitor against phosphorylation of the MAPKs were also evaluated. IL-6 and CXCL8 release by Pa were significantly inhibited by p38MAPK and Syk inhibitors (p<0.05). Phosphorylation of HSP27, but not ERK or JNK, was significantly inhibited by Syk kinase inhibition. A combination of p38MAPK and Syk inhibitors showed synergy against IL-6 and CXCL8 induction and an NSKI completely inhibited IL-6 and CXCL8 at low concentrations. Pa-induced inflammation is dependent on p38MAPK primarily, and Syk partially, which is upstream of p38MAPK. The NSKI suggests that inhibiting specific combinations of kinases is a potent potential therapy for Pa-induced inflammation.

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4

Wang, Xing, Junfang Guo, Zhongqi Ning, and Xia Wu. "Discovery of a Natural Syk Inhibitor from Chinese Medicine through a Docking-Based Virtual Screening and Biological Assay Study." Molecules 23, no. 12 (November 28, 2018): 3114. http://dx.doi.org/10.3390/molecules23123114.

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Spleen tyrosine kinase (Syk) is a critical target protein for treating immunoreceptor signalling-mediated allergies. In this study, a virtual screening of an in-house Chinese medicine database followed by biological assays was carried out to identify novel Syk inhibitors. A molecular docking method was employed to screen for compounds with potential Syk inhibitory activity. Then, an in vitro kinase inhibition assay was performed to verify the Syk inhibitory activity of the virtual screening hits. Subsequently, a β-hexosaminidase release assay was conducted to evaluate the anti-mast cell degranulation activity of the active compounds. Finally, tanshinone I was confirmed as a Syk inhibitor (IC50 = 1.64 μM) and exhibited anti-mast cell degranulation activity in vitro (IC50 = 2.76 μM). Docking studies showed that Pro455, Gln462, Leu377, and Lys458 were key amino acid residues for Syk inhibitory activity. This study demonstrated that tanshinone I is a Syk inhibitor with mast cell degranulation inhibitory activity. Tanshinone I may be a potential lead compound for developing effective and safe Syk-inhibiting drugs.

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5

Bertoni, Francesco, Andrea Rinaldi, Anna Sasso, Gianluca Gaidano, Emanuele Zucca, Silvia Uccella, Giancarlo Pruneri, et al. "In Vitro Activity of SYK and BCR-ABL Inhibitors in Aggressive Lymphomas." Blood 108, no. 11 (November 16, 2006): 2520. http://dx.doi.org/10.1182/blood.v108.11.2520.2520.

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Abstract The B cell receptor tyrosine kinase SYK is a critical component of the B-cell receptor signalling pathway in normal B cells. We have recently reported that SYK is amplified and over-expressed in mantle cell lymphoma (MCL) and that the growth of MCL and diffuse large B cell lymphoma (DLBCL) cell lines over-expressing SYK is inhibited by piceatannol, a known SYK inhibitor (Bertoni et al, ASH 2005; Rinaldi et al, BJH 2006). Others have reported important SYK expression in splenic marginal zone B cell lymphomas, in DLBCL and peripheral T cell lymphomas (PTCL) (Ruiz-Ballesteros et al, 2005; Mahadevan et al, w Streubel et al, 2006), suggesting SYK targeting agents could be useful for the treatment of various lymphoma subtypes. SYK inhibitors are already in clinical development for treatment of asthma. Here, we report on the activity on lymphoma cell lines and primary cells of the SYK/ZAP-70 inhibitor #1 (Novartis) and of the BCR-ABL inhibitors imatinib (Novartis) and nilotinib (Novartis), which could act as cross-selecting SYK inhibitors (Atwell et al, 2004). We treated four human MCL and three DLBCL established cell lines with increasing doses of the SYK/ZAP-70 inhibitor #1, imatinib and nilotinib for 72 h. Cell viability was measured with the MTT assay. The two cell lines expressing high levels of SYK, JeKo-1 and SUD-HL-6, were sensitive to the compounds (IC50: Syk/ZAP-70 inhibitor #1, 1–5 μM; imatinib, 15–20 μM; nilotinib, 10 μM). Cells with lower SYK expression were generally less sensitive to all three compounds. To obtain further data on the relevance of SYK inhibition in lymphoma, we treated nine lymphoma primary cells with the piceatannol (Sigma), the SYK/ZAP-70 inhibitor #1 and nilotinib. Responses, defined as <50% decrease in viable cell number, were observed with piceatannol (4/9 samples), the Syk/ZAP-70 inhibitor #1 (3/9 samples) and nilotinib (3/9 samples). Immunoblotting experiments aimed to elucidate the mechanism of action are under-way and data will be presented at the meeting. In conclusion, our data indicate that pharmacological inhibition of SYK is a therapeutic approach to be further investigated in subsets of aggressive lymphomas.

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6

Buchner, Maike, Simon Fuchs, Gabriele Prinz, Dietmar Pfeifer, Kilian Bartholomé, Nina Chevalier, Laurent Vallat, et al. "Spleen Tyrosine Kinase (SYK) Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia." Blood 112, no. 11 (November 16, 2008): 543. http://dx.doi.org/10.1182/blood.v112.11.543.543.

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Abstract B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is characterized by expansion of monoclonal mature B lymphocytes. Despite advances in treatment, the disease remains incurable warranting further efforts to identify novel molecular targets in CLL. B cell receptor (BCR) signaling contributes to apoptosis resistance in CLL limiting the efficacy of therapeutic approaches. In this study we investigated the expression of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared to healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCγ2, STAT3, and ERK1/2 in CLL compared to healthy B cells suggesting enhanced activation of these mediators in CLL. Separate analyses according to prognostic parameters revealed 1.8-fold higher SYK protein level in unmutated compared to mutated CLL cells determined by densitometric analysis (n=22, p=0.0031). These findings may well explain the higher BCR signaling capacity in the unmutated CLL subset. Various SYK inhibitors (piceatannol, curcumin, SYK Inhibitor II, and IV (Calbiochem)) reduced phosphorylation of the SYK downstream targets PLCγ2, STAT3, and ERK1/2 in a time- and dose-dependent manner and induced apoptosis in the CLL cell lines Mec-1 and EHEB and primary CLL cells. SYK Inhibitor II revealed highest cytotoxic effects on primary CLL cells, but did not significantly reduce the viability of healthy B cells. Thus, apoptotic effects of this inhibitor were analyzed in a larger cohort of patient samples along with the well-established SYK inhibitor R406 (Rigel Inc.). After 48 hour treatment, relative viability of CLL cells was reduced to 76% and 44% for SYK Inhibitor II (4 mM and 10 mM) and to 66% for R406 (4 mM), respectively (n=38, p<0.0001). With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70+ cases. Cytotoxicity of SYK inhibitors were associated with SYK protein expression potentially predicting response to therapy (Pearson correlation coefficient: r=0.78 for SYK Inhibitor II (p<0.0001) and r=0.56 for R406 (p=0.0134)). Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared to fludarabine therapy alone. The mean viability of F-ara-A treated cells (10 mM) was reduced by 13% with SYK II and by 17% with R406 (n=10, p<0.0001), respectively. Since microenvironment enhances the viability of CLL cells and decreases their sensitivity towards chemotherapy, apoptosis rates of CLL cells incubated with SYK Inhibitor II in the presence and absence of the stromal cell line M2-10B4 were assayed. No significant change in cytotoxic effects was observed. Hence, stromal cell interactions do not impair the cytotoxicity of SYK inhibitors. Moreover, CD40L expressing T cells play an important role in the CLL microenvironment, and CD40 ligation is under discussion to induce fludarabine resistance in CLL. Therefore the effect of SYK Inhibitor II in combination with CD40 ligation was analyzed. In contrast to conventional chemotherapy, stimulation with CD40L significantly sensitized CLL cells towards SYK inhibition. In conclusion, this work establishes SYK inhibition as a rational and promising therapeutic principle in CLL. Given the preferential activity of SYK inhibitors in CLL cases with poor prognostic factors, the independence of their activity from the protective influence of the CLL microenvironment, and the synergistic and complementary action with fludarabine, we propose SYK inhibitors for clinical assessment in the therapy of CLL.

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Yang, Na, Wei Deng, Qiaoling Sun, Junqing Liang, Linfang Wang, Shiming Fan, Renxiang Tang, et al. "HMPL-523, a Novel SYK Inhibitor Showed Anti-Tumor Activities In Vitro and In Vivo." Blood 128, no. 22 (December 2, 2016): 3970. http://dx.doi.org/10.1182/blood.v128.22.3970.3970.

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Abstract Introduction: Spleen Tyrosine Kinase (SYK), a non-receptor type of tyrosine kinase, is a member of Syk/ZAP70 tyrosine kinase family. It plays a pivotal role in the regulation of B-cell receptor (BCR) signal pathway, which regulates proliferation, differentiation and survival of B lymphocytes. The abnormal activation of BCR singling is closely related to transformation and development of B cell lymphoma. Targeting BCR downstream molecules, such as Bruton' tyrosine kinase (BTK) and phosphoinositide-3-kinase δ (PI3Kδ) has emerged as new therapeutic approaches and inhibitors of BTK and PI3Kδ were approved recently by FDA for treatment of some subtypes of B-cell malignancies. Currently, a couple small molecular inhibitors against SYK, another BCR downstream molecule, are under the early clinical development and showed initial efficacy in B cell lymphomas. HMPL-523, discovered and currently being developed in Phase I clinical trial by Hutchison MediPharma, is a novel, highly potent and selective SYK inhibitor (IC50: 0.025 μM). The anti-tumor activity of HMPL-523 was evaluated in this study. Methods: Inhibitory effects of HMPL-523 on cell viability were investigated in a panel of B cell lymphoma cell lines with SYK/BCR dysregulation by CellTiter-Glo luminescent or CCK-8 assay. The effect of HMPL-523 on SYK signaling pathway was detected by western blot. Annexin-V- positive and PI-negative population was recognized as apoptotic cells by FACS. Nude mice bearing B cell lymphoma xenograft tumors with SYK/BCR dysregulation were used to determine anti-tumor activity of HMPL-523 in vivo. Result: HMPL-523 blocked phosphorylation of BLNK, downstream protein of Syk, in human mantle cell line REC-1 and human plasma cell line ARH-7777 with IC50 of 0.105 µM and 0.173 μM, respectively. HMPL-523 also inhibited cell viability of Ba/F3 Tel-Syk with IC50 of 0.033 μM. Furthermore, inhibitory effects of HMPL-523 on cell viability were evaluated in a panel of B -cell lymphoma cell lines with SYK/BCR deregulation. Results showed that HMPL- 523 potently inhibited cell survival with IC50s from 0.4 to 2 μM. Consistent with the effect on cell viability, HMPL-523 increased the apoptotic rate of REC-1 cells. Moreover, HMPL-523 showed the synergistic activities on killing human diffused large B cell lymphoma (DLBCL) in combination with other drugs such as BTK inhibitor, PI3Kδ inhibitors and Bcl2 family inhibitor. The detailed mechanisms underlying the synergism are still under investigation. Anti-tumor activity of HMPL-523 was determined in Syk dependent xenograft models. Daily oral administration of 100 mg/kg HMPL-523 showed potent anti-tumor activity in B cell lymphoma REC-1 (TGI: 59%). Conclusion:HMPL-523 is a highly potent SYK inhibitor with good activity against B-cell lymphoma in pre-clinical in vitro and in vivo models, supporting further clinical research for HMPL-523 as either single agent or combination drug with other agents to treat B-cell malignancies e.g. DLBCL Disclosures Yang: Hutchison MediPharma Ltd: Employment, Research Funding. Deng:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Research Funding. Liang:Hutchison MediPharma Ltd: Employment, Research Funding. Wang:Hutchison MediPharma Ltd: Employment, Research Funding. Fan:Hutchison MediPharma Ltd: Employment, Research Funding. Tang:Hutchison MediPharma Ltd: Employment, Research Funding. Yu:Hutchison MediPharma Ltd: Employment, Research Funding. Sun:Hutchison MediPharma Ltd: Employment, Equity Ownership. Zhou:Hutchison MediPharma Ltd: Employment, Research Funding. Dai:Hutchison MediPharma Ltd: Employment, Research Funding. Qing:Hutchison MediPharma Ltd: Employment, Research Funding. Su:Hutchison MediPharma Ltd: Employment, Research Funding. Ren:Hutchison MediPharma Ltd: Employment, Research Funding.

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Huang, Duen-Yi, Wei-Yu Chen, Chi-Long Chen, Nan-Lin Wu, and Wan-Wan Lin. "Synergistic Anti-Tumour Effect of Syk Inhibitor and Olaparib in Squamous Cell Carcinoma: Roles of Syk in EGFR Signalling and PARP1 Activation." Cancers 12, no. 2 (February 19, 2020): 489. http://dx.doi.org/10.3390/cancers12020489.

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Syk is a non-receptor tyrosine kinase involved in the signalling of immunoreceptors and growth factor receptors. Previously, we reported that Syk mediates epidermal growth factor receptor (EGFR) signalling and plays a negative role in the terminal differentiation of keratinocytes. To understand whether Syk is a potential therapeutic target of cancer cells, we further elucidated the role of Syk in disease progression of squamous cell carcinoma (SCC), which is highly associated with EGFR overactivation, and determined the combined effects of Syk and PARP1 inhibitors on SCC viability. We found that pharmacological inhibition of Syk could attenuate the EGF-induced phosphorylation of EGFR, JNK, p38 MAPK, STAT1, and STAT3 in A431, CAL27 and SAS cells. In addition, EGF could induce a Syk-dependent IL-8 gene and protein expression in SCC. Confocal microscopic data demonstrated the ability of the Syk inhibitor to change the subcellular distribution patterns of EGFR after EGF treatment in A431 and SAS cells. Moreover, according to Kaplan-Meier survival curve analysis, higher Syk expression is correlated with poorer patient survival rate and prognosis. Notably, both Syk and EGFR inhibitors could induce PARP activation, and synergistic cytotoxic actions were observed in SCC cells upon the combined treatment of the PARP1 inhibitor olaparib with Syk or the EGFR inhibitor. Collectively, we reported Syk as an important signalling molecule downstream of EGFR that plays crucial roles in SCC development. Combining Syk and PARP inhibition may represent an alternative therapeutic strategy for treating SCC.

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Getz, Todd M., Bhanu Manne, Lorena Buitrago, Yingying Mao, and Satya P. Kunapuli. "Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets." Thrombosis and Haemostasis 109, no. 06 (2013): 1131–40. http://dx.doi.org/10.1160/th12-09-0645.

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SummaryIn our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

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Issara-Amphorn, Jiraphorn, Naraporn Somboonna, Prapaporn Pisitkun, Nattiya Hirankarn, and Asada Leelahavanichkul. "Syk inhibitor attenuates inflammation in lupus mice from FcgRIIb deficiency but not in pristane induction: the influence of lupus pathogenesis on the therapeutic effect." Lupus 29, no. 10 (July 22, 2020): 1248–62. http://dx.doi.org/10.1177/0961203320941106.

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Macrophages are responsible for the recognition of pathogen molecules. The downstream signalling of the innate immune responses against pathogen molecules, lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG), and the adaptive immune response to antibodies, Fc gamma receptor (FcgR), is spleen tyrosine kinase (Syk). Because pathogen molecules and antibodies could be presented in lupus, impact of Syk and macrophages in lupus is explored. FcgR-IIb deficient (FcgRIIb-/-) mice, a model of inhibitory signalling loss, at 40 weeks old, but not pristane mice (a chemical induction lupus model) demonstrated spontaneous elevation of LPS and BG in serum from gut translocation despite the similarity in faecal microbiome analysis. Syk abundance in FcgRIIb–/– mice was higher than in pristane mice, possibly due to several Syk activators (anti-dsDNA, LPS and BG), and Syk inhibitor–attenuated proteinuria and serum cytokines only in FcgRIIb–/– mice. In addition, LPS + BG enhanced the expression of activating FcgRs, NF-κB and Syk, together with supernatant TNF-α predominantly in FcgRIIb–/– compared to wild-type macrophages. The inhibitors against Dectin-1, Syk and nuclear factor kappa B, but not anti-Raf-1, reduced supernatant TNF-α in LPS+BG-activated macrophages, implying Syk-dependent signalling. The pathogen molecules enhanced activating-FcgRs, without inhibition, through Syk, a shared downstream innate and adaptive signalling, is responsible for the hyper-responsiveness in FcgRIIb–/– macrophages. In conclusion, Syk inhibitor attenuated inflammation in FcgRIIb–/– but not in pristane mice, implying the influence of a lupus genetic background in treatment modalities.

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Raeder, Evelin M. B., Pamela J. Mansfield, Vania Hinkovska-Galcheva, James A. Shayman, and Laurence A. Boxer. "Syk Activation Initiates Downstream Signaling Events During Human Polymorphonuclear Leukocyte Phagocytosis." Journal of Immunology 163, no. 12 (December 15, 1999): 6785–93. http://dx.doi.org/10.4049/jimmunol.163.12.6785.

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Abstract We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cδ (PKCδ) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCδ and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCδ and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcγ receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.

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Kiliszek, Przemyslaw, Maciej Szydlowski, Tomasz Sewastianik, Ewa Jablonska, Emilia Bialopiotrowicz, Patryk Gorniak, Anna Polak, et al. "FOXO1 Activation Is an Effector of SYK and AKT Inhibition in Tonic BCR Signal-Dependent Diffuse Large B-Cell Lymphomas." Blood 126, no. 23 (December 3, 2015): 314. http://dx.doi.org/10.1182/blood.v126.23.314.314.

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Abstract Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. Herein, we investigated the role of FOXO1 in toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas. Methods: BCR-dependent DLBCL cell lines (DHL4, DHL6, Ly1, Ly7) were incubated with SYK inhibitor R406 (4 µM) and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. Toxicity of active FOXO1 was assessed by transducing DLBCL cells with a constitutively nuclear FOXO1 mutant. FOXO1 knock-down in DLBCL cell was achieved with shRNA. DREAM cleavage and HRK expression were assessed by Western Blot and qPCR, respectively, in the absence or presence of caspase inhibitors. DLBCL cell viability and apoptosis after incubation with SYK and/or AKT inhibitors R406 and MK2206, were assessed by MTS assay and Annexin V/PI staining. Drug interactions were quantitated by CompuSyn software. The expression of p-SYK and FOXO1 in DLBCL samples was assessed by immunohistochemistry (IHC) in 60 DLBCL patients. Results: Since FOXO1 is a major effector of tonic BCR signaling, we assessed the activity of FOXO1 in DLBCL cells after SYK inhibition. In all tested cell lines, AKT and FOXO1 phosphorylations decreased after incubation with SYK inhibitor. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent expression of p27, BIM, TRAIL and GADD45A. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1-3A mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, whereas wild-type FOXO1 did not change proliferation or cellular viability, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with depleted FOXO1 exhibited 70% lower sensitivity to SYK inhibitor than control cells (p<0.0001). Since in previous studies expression of the proapoptotic member of BCL2 family, HRK, was required for SYK-inhibitor induced cell death in DLBCL cells, we determined the role of FOXO1 activation in HRK expression. HRK expression was dramatically increased in SYK-inhibitor treated control cells, but not in FOXO1-deficient cells. Consistent with this, SYK inhibitor triggered cleavage of HRK transcriptional repressor DREAM only in control cells, but not in FOXO1-depleted cells. HRK induction was blocked by caspase inhibitors. Since AKT is major kinase regulating both FOXO1 activity and HRK induction, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic toxicity (combination index [CI] 0.5-0.8). Combination of inhibitors in FOXO1-depleted cells did not trigger cell death, highlighting the critical effector role of FOXO1. FOXO1 expression was present in 80% of primary DLBCL samples and correlated with SYK activity (p=0.009). High levels of FOXO1 protein expression were associated with longer OS (log rank, p=0.04). Conclusions: Taken together, our results demonstrate that targeting the BCR signaling at multiple levels is a rational therapeutic strategy. Proapoptotic activity of FOXO1 is an important effector of SYK and AKT inhibition in DLBCLs and its expression is required for SYK-and AKT inhibitor-induced toxicity. The underlying mechanism linking FOXO1 activation and cell death involves caspase-dependent cleavage of transcriptional repressor DREAM and subsequent induction of a proapoptotic BCL2 family member, HRK. Disclosures No relevant conflicts of interest to declare.

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Issara-Amphorn, Jiraphorn, Wiwat Chancharoenthana, Peerapat Visitchanakun, and Asada Leelahavanichkul. "Syk Inhibitor Attenuates Polymicrobial Sepsis in FcgRIIb-Deficient Lupus Mouse Model, the Impact of Lupus Characteristics in Sepsis." Journal of Innate Immunity 12, no. 6 (2020): 461–79. http://dx.doi.org/10.1159/000509111.

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The impact of spleen tyrosine kinase (Syk) signaling might be prominent in lupus because (i) Syk is a shared downstream signaling molecule among circulating immune complex, LPS, and (1→3)-β-D-glucan (BG), and (ii) all of these factors are detectable in the serum of Fc gamma receptor IIb-deficient (FcgRIIb−/−) mice with sepsis. As a proof of concept study, we activated macrophages with BG combined with LPS (BG + LPS). We found that BG + LPS predominantly upregulated Syk expression and proinflammatory cytokines in FcgRIIb−/− macrophages compared with wild-type (WT) macrophages. Syk inhibition downregulated several inflammatory pathways in FcgRIIb−/− macrophages activated with BG + LPS, as determined by RNA sequencing analysis, suggesting the potential anti-inflammatory impact of Syk inhibitors in lupus. Indeed, administration of a Syk inhibitor prior to cecal ligation and puncture (CLP) sepsis in FcgRIIb−/− mice reduced baseline lupus-induced proinflammatory cytokines and attenuated sepsis severity as evaluated by mortality, organ injury, serum LPS, and post-sepsis serum cytokines. In conclusion, it was easier to induce Syk expression in FcgRIIb−/− macrophages than in WT macrophages. This might be because of the loss of inhibitory signaling, which might be responsible for prominent Syk abundance in the spleens of 40-week-old FcgRIIb−/− mice and the potent effect of Syk inhibitor in lupus mice compared with WT.

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Chen, Linfeng, Przemyslaw Juszczynski, Yasumichi Hitoshi, Wen Chen, Jeffery L. Kutok, and Margaret A. Shipp. "Tonic B-Cell Receptor Signaling Promotes the Survival of Diffuse Large B-Cell Lymphomas: Identification of SYK as a Rational Treatment Target." Blood 108, no. 11 (November 16, 2006): 226. http://dx.doi.org/10.1182/blood.v108.11.226.226.

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Abstract The role of BCR-mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. B-cell antigen receptor (BCR) signaling induces receptor oligomerization and Ig alpha/beta ITAM phosphorylation. Thereafter, the protein tyrosine kinase (PTK) SYK is recruited and activated, initiating downstream events and amplifying the original BCR signal. Recent studies indicate that BCRs also transmit low-level survival signals in the absence of receptor engagement and highlight the critical role of SYK in this “tonic” BCR signaling. We recently found that SYK is a major substrate of the protein tyrosine phosphatase, PTPROt. PTPROt-mediated dephosphorylation and inactivation of SYK inhibited DLBCL proliferation and induced apoptosis in the absence of BCR cross-linking. These observations suggested that tonic BCR signaling may be an important survival mechanism in certain DLBCLs and identified SYK as a possible rational therapeutic target. For these reasons, we assessed the efficacy of a recently developed ATP-competitive inhibitor of SYK, R406, in a panel of 8 DLBCL cell lines. R406 inhibited the proliferation of 6 DLBCL cell lines at IC50s of 0.9–1.8 micromolar. The SYK inhibitor was cytotoxic (30–85% apoptotic cells) at low micromolar doses in 6 of these lines. To delineate the relationship between tonic BCR signaling, associated SYK activity and R406 responsiveness, we assessed SYK phosphorylation (pTYR 352 and 525/526) in the DLBCL cell line panel. Upon BCR signaling, SYK is phosphorylated at TYR 352; thereafter, the kinase undergoes autophosphorylation at TYR 525/526, which is required for SYK activity. In sensitive DLBCL cell lines, R406 specifically inhibited TYR 525/526 phosphorylation and additional downstream signals (p-ERK). In addition, all 6 R406-sensitive DLBCL cell lines exhibited basal SYK 352 phosphorylation whereas the 2 R406-resistant lines lacked pTyr 352 phosphorylation. Basal SYK pTYR 352 could be detected by immunoblotting total SYK immunoprecipitates with a phospho-specific SYK 352 antibody or using the same pTYR 352 antibody for intracellular phospho-specific flow cytometry. We conclude that :tonic BCR signaling is an important survival mechanism in many DLBCLs;SYK is a rational therapeutic target in these tumors;R406 is a promising SYK inhibitor; andSYK pTYR 352 is an excellent indicator of tonic BCR signaling and predictor of R406 sensitivity in DLBCL.

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Chen, Liguang, George Chen, Jessie-Farah Fecteau, Greg Coffey, Charles Prussak, Dennis A. Carson, and Thomas Kipps. "Highly Specific Inhibitor for Syk Induces Chronic Lymphocytic Leukemia Cell Apoptosis,." Blood 118, no. 21 (November 18, 2011): 3875. http://dx.doi.org/10.1182/blood.v118.21.3875.3875.

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Abstract Abstract 3875 The antibodies expressed by CLL cells are highly selected, implicating a role for antigen in the pathogenesis of this disease. The B-cell receptor (BCR) for antigen may become engaged by microbial or self-antigens to induce activation of downstream adapter/signaling molecules. This, either alone or in concert with activation of other receptors, can induce CLL-cell expression of genes encoding proteins involved in cell survival or proliferation, e.g. c-MYC, potentially enhancing the tendency for disease progression. Our prior studies found that ZAP-70 in CLL cells enhanced BCR signaling, which might account for the relatively aggressive clinical behavior of patients who have CLL cells that express this protein. Although transduction of ZAP-70-negative CLL cells with vectors encoding ZAP-70 enhanced BCR signaling, we found this effect did not require an active ZAP-70 kinase domain. Instead ZAP-70 apparently acts as an adaptor protein that facilitates recruitment/activation of Syk, which in turn serves as the principal kinase responsible for initiating BCR signaling. As such, inhibitors of Syk could be useful therapeutically, as indicated by a clinical study demonstrating that one Syk inhibitor, fostamatinib disodium (R406/R788), has activity in the treatment of patients with this disease. However, at concentrations of fostamatinib required for clinical activity this drug also can inhibit several kinases other than Syk, including Lck, JAK, and Flt3, making it difficult to conclude that the activity of this drug solely was due to its capacity to inhibit Syk. Coffey and colleagues developed a series of highly potent and selective small molecule Syk inhibitors. One of these inhibitors, PRT060318, is a highly specific inhibitor of Syk that is more selective than fostamatinib. We examined whether this specific Syk inhibitor could affect BCR signaling and CLL-cell survival. We evaluated the effect of PRT060318 on BCR signaling by calcium influx induced through surface IgM ligation. We found that PRT060318 inhibited IgM induced BCR signaling with an IC50∼200nM. CLL cells that express ZAP-70 (n=13) or that lacked expression of this kinase (n=10) were treated with 5 μM PRT060318 and examined for cell viability 24 hours later. The average viability of CLL cells that expressed ZAP-70 was significantly lower (70.4 ± 16.1) than that of CLL cells that lacked expression of ZAP-70 (93.6 ± 9.0) (P<0.01), revealing that PRT060318 had greater activity in inducing apoptosis of CLL cells that had high-level expression of ZAP-70. Moreover, Syk inhibition abrogated the capacity of surface IgM ligation to enhance CLL cell survival, the viability of CLL cells treated with PRT060318 and anti-μ (38.8 ± 5.4) was significantly lower than CLL cells with anti-μ stimulation after 48 hours (64.0 ± 6.0) (P<0.01, n=3). PRT060318 also inhibited the capacity of marrow stromal cells to support the survival of CLL cells in vitro (35.3 ± 5.2 vs. 61.8 ± 4.7) (P<0.01, n=4). These results indicate that selective Syk inhibition by PRT060318 can induce apoptosis in CLL cells, has greater activity against CLL cells that express ZAP-70 and might be most active in treating patients with aggressive disease characteristics. Disclosures: Coffey: Portola Pharmaceuticals Inc.: Employment. Carson:Wintherix: Equity Ownership. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Research Funding; Amgen: Research Funding.

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Han, Yingjie, Frank Y. Ma, Julie Di Paolo, and David J. Nikolic-Paterson. "An inhibitor of spleen tyrosine kinase suppresses experimental crescentic glomerulonephritis." International Journal of Immunopathology and Pharmacology 32 (January 1, 2018): 205873841878340. http://dx.doi.org/10.1177/2058738418783404.

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Non-selective inhibitors of spleen tyrosine kinase (SYK) efficiently suppress disease in T cell-dependent models of crescentic glomerulonephritis. However, the therapeutic potential of selective SYK inhibitors in this disease has not been established. In addition, we lack knowledge regarding SYK expression in non-myeloid cells in glomerulonephritis. We addressed these two issues in a rat model of nephrotoxic serum nephritis (NTN) using a SYK inhibitor, GS-492429. Disease was induced in Sprague-Dawley rats (Study 1) or Wistar-Kyoto (WKY) rats (Study 2) by immunization with sheep IgG and administration of sheep anti-rat nephrotoxic serum. Animals were untreated or received GS-492429 (30 mg/kg/bid) or vehicle treatment from 2 h before nephrotoxic serum injection until being killed 3 or 24 h later (Study 1) or 14 days later (Study 2). Two-colour confocal microscopy found that SYK expression in NTN kidney was restricted to myeloid cells and platelets, with no evidence of SYK expression by T cells, mesangial cells, podocytes or tubular epithelial cells. In Study 1, GS-492429 treatment significantly reduced glomerular neutrophil and macrophage infiltration, with protection from glomerular thrombosis and proteinuria. In Study 2, GS-492429 treatment reduced glomerular crescent formation by 70% on day 14 NTN in conjunction with reduced glomerular thrombosis, glomerulosclerosis and tubular damage. This was accompanied by a marked reduction in markers of inflammation (CCL2, TNF-α, NOS2, MMP-12). Importantly, the protective effects of GS-492429 were independent of T cell infiltration and activation and independent of JAK/STAT3 signalling. In conclusion, this study demonstrates that a SYK inhibitor can suppress the development of crescentic glomerulonephritis through effects upon myeloid cells and platelets.

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Abraham, Shaji, Leonard C. Edelstein, Chad A. Shaw, Pierrette Andre, Xianguo Kong, Carol T. Dangelmaier, Alexander Tsygankov, Paul F. Bray, Satya P. Kunapuli та Steven E. McKenzie. "Platelet FcγRIIA Signaling Results in Ubiquitination and Cellular Translocation of Activated Syk". Blood 124, № 21 (6 грудня 2014): 2761. http://dx.doi.org/10.1182/blood.v124.21.2761.2761.

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Abstract Platelet FcγRIIA is central to the pathophysiology of immune-mediated thrombocytopenia and thrombosis syndromes, such as heparin-induced thrombocytopenia (HIT). FcγRIIA is also the major transmembrane signaling adapter for αIIbβ3 outside-in signaling. In HIT, antibody to heparin/PF4 is necessary but not sufficient for disease to occur. Inter-individual variation in platelet activation via FcγRIIA contributes to HIT risk, but the molecular basis for the variation is incompletely understood. In our PRAX1 study of platelet reactivity and RNA expression (Edelstein, Nature Med 2013; Simon, Blood 2014), we identified differentially expressed mRNAs from healthy donors with different platelet reactivity to FcγRIIA stimulation. We observed significant differential expression of molecules involved in ubiquitination processes in relation to platelet reactivity to FcγRIIA stimulation. Syk is a protein tyrosine kinase and the major signaling node downstream of platelet receptors that use immunotyrosine activation motif (ITAM) signaling, such as FcγRIIA, GPVI and CLEC-2. We previously reported Syk ubiquitination following GPVI stimulation, and the role of c-cbl as the E3 ubiquitin ligase (Dangelmaier, Blood 2005). Ubiquitination is an important post-translational modification that modulates signal transduction by regulating the activity, subcellular localization or stability of proteins. We tested the hypothesis that ubiquitination participates in signaling, and examined ubiquitination of Syk downstream of platelet FcγRIIA activation. Using both washed human platelets and HEL cells, we observed ubiquitination of Syk upon FcγRIIA engagement by cross-linking IV.3 mAb (10 ug/ml) with goat anti-mouse Fab’2 (30 ug/ml). Both tyrosine phosphorylation and ubiquitination of Syk occurred within 15 sec, peaked by 1-3 min and decreased thereafter. The pattern of ubiquitination was consistent with 1 to 3 Ub molecules per Syk molecule. Ubiquitinated-Syk (Ub-Syk) was increased in the presence of PR-619, a deubiquitinating enzyme inhibitor, confirming ubiquitination of Syk. Ub-Syk associates with the cytoskeletal-rich platelet fraction, membrane skeleton fraction and with cytosolic fraction in detergent lysed platelets that were fractionated by lower g-forces (15,500 x g) and higher g-forces (100,000 x g). This suggests that Ub-Syk is translocated into all cellular compartments upon platelet activation. Ub-Syk was absent upon pre-treatment with Src-family kinase inhibitor PP2 (10 uM), but minimally affected in the presence of Syk inhibitor PRT318 (1 uM), in both platelets and HEL cells, as compared to DMSO treated control cells. Further, phosphorylation of c-cbl was inhibited strongly by PP2, but only slightly inhibited by PRT318, suggesting that ubiquitination of Syk depends on Src kinase activity. Of note, Ub-Syk was not degraded by the proteasome, since no accumulation of Ub-Syk was observed by pretreatment with proteasome inhibitors MG132 or Epoxomicin in either platelets or HEL cells, compared to control cells. In conclusion, Syk is ubiquitinated upon cross-linking platelet FcγRIIA and is translocated to all major subcellular compartments. Since ubiquitinated Syk is activation-dependent and not subject to proteasomal degradation, it likely serves as a novel adapter molecule for protein-protein interactions in mediating platelet activation via FcγRIIA. Disclosures No relevant conflicts of interest to declare.

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Zhou, Zhou, Francisca C. Gushiken, Angela Bergeron, Vinod K. Vijayan, Rolando Rumbaut, Jose A. Lopez, and Jing-fei Dong. "STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of Its Transcription Factor Activity." Blood 116, no. 21 (November 19, 2010): 157. http://dx.doi.org/10.1182/blood.v116.21.157.157.

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Abstract Abstract 157 Signal Transducer and Activator of Transcription 3 (STAT3) serves as a transcription factor activated by cytokine-induced intracellular signals, which are critical in megakaryopoiesis. This signaling pathway may also be active in anucleated platelets that are primed by proinflammatory cytokines, suggesting that STAT3 plays a role in platelet hyperactivity associated with inflammation. We have recently found that three different classes of STAT3 inhibitors each selectively inhibited collagen-induced aggregation of human platelets by ∼50%. They also blocked thrombus formation (∼80%) on immobilized collagen under an arterial shear stress of 62.5 dyn/cm2. These STAT3 inhibitors also blocked platelet aggregation induced by collagen-related peptide, suggesting that they acted on GP VI-mediated intracellular signaling in platelets. These in vitro results were further verified in two sets of experiments in mouse models. First, an oligonucleotide G-quartet STAT3 inhibitor (1 mg/ml) or a scrambled control oligonucleotide were infused into C57/BJ6 mice daily for three days. Collagen-induced platelet aggregation was then induced and found to be reduced by up to 60% in mice infused with the STAT3 inhibitor, but not with the control oligonucleotide. Photochemical injury-induced thrombosis in the cremaster arterioles was also significantly delayed in the inhibitor-infused mice as compared to control mice. Second, infusing STAT3 inhibitor could result in platelet inhibitor indirectly by acting endothelial cells. To address this concern, we have generated platelet-specific STAT3 null mice that have developed normally and have normal platelet counts. The collagen-, but not TRAP-induced platelet aggregation in the platelet STAT3 KO mice was reduced as compared to their littermates. Platelets from the platelet-specific STAT3 KO mice were also significantly defective in thrombus formation on immobilized collagen under 62.5 dyn/cm2 of fluid shear stress that was generated in a parallel-plate flow chamber system. Consistent with results from these functional assays, collagen induced rapid (peaked at 5 min after stimulation) and dose-dependent tyrosine phosphorylation of STAT3, but not of STAT1 or STAT5 in washed human platelets. The phosphorylation was blocked dose-dependently by two STAT3 inhibitors. Syk inhibitors also blocked collagen-induced STAT3 phosphorylation in a dose-dependent manner, but STAT3 inhibitors had no effect on Syk phosphorylation, suggesting that Syk acts upstream of STAT3. Furthermore, STAT3 inhibitors also dose-dependently reduced collagen-induced tyrosine phosphorylation of PLCγ2, which is a known substrate of Syk. Consistent with this temporal interaction among STAT3, Syk and PLCγ2, activated STAT3 co-immunoprecipitated phosphorylated Syk and PLCγ2 in collagen-activated human platelets. The tri-molecular complex was also immunoprecipitated by an antibody to PLCγ2. Taken together, these data suggest that STAT3 regulates collagen-induced platelet aggregation, independent of its transcription factor activity. The regulation is potentially achieved by STAT3 serving as a protein scaffold linking the kinase Syk with its substrate PLCγ2 to enhance the signal relay in collagen-activated platelets. This cross-talk between collagen and cytokine signaling pathways provides a mechanism for how proinflammatory mediators could prime platelets for activation by hemostatic ligands. Disclosures: No relevant conflicts of interest to declare.

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Abraham, Shaji, Carol T. Dangelmaier, Yuhang Zhou, Leonard C. Edelstein, Paul F. Bray, Satya P. Kunapuli та Steven E. McKenzie. "Syk Is Regulated Downstream Of FcγRIIA In Platelets By Transient Tyrosine Phosphorylation and Ubiquitylation". Blood 122, № 21 (15 листопада 2013): 4737. http://dx.doi.org/10.1182/blood.v122.21.4737.4737.

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Platelet FcγRIIA contributes to the pathophysiology of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis syndromes. Activation of FcγRIIA results in tyrosine phosphorylation of Syk in human platelets, but little is known about ubiquitylation of Syk. Protein ubiquitylation has been shown to regulate physiological and pathological cellular processes by regulating signaling networks. In the Platelet RNA and Expression-1 (PRAX1) study, we studied platelets from 154 healthy subjects and identified increased expression of mRNAs encoding proteins involved in ubiquitylation in platelets that were highly reactive to FcγRIIA stimulation. Previously, we (CD, SPK) reported that platelet stimulation via GPVI/FcRγ results in transient Syk phosphorylation and ubiquitylation, involving c-cbl and TULA-2. Briefly, c-Cbl acts as an E3 ligase transferring ubiquitin to the lysine residue of the target protein in the ubiquitylation reaction and TULA-2 is a dephosphorylating enzyme that removes phosphate group from ubiquitinated syk. In this study, we investigated Syk tyrosine phosphorylation and ubiquitylation downstream of platelet FcγRIIA engagement. FcγRIIA on washed human platelets was activated by cross-linking with IV.3 antibody (10ug/ml) and goat anti-mouse antibody (GAM) (30ug/ml). At specific time intervals following activation, platelets were lysed and the lysates were immunoblotted for total Syk and for pY525/526 phospho-Syk. We observed phosphorylation and ubiquitylation of Syk within 15 sec, and peaking within 1 to 3 min. The MW of the Syk species was consistent with 1 to 3 ubiquitin (Ub) moieties per Syk molecule. Alternatively, platelet FcγRIIA was activated via anti-CD9; Ub-Syk was observed at 15 sec, while ubiqutinated and phosphorylated Syk was observed at 2 min. Additional ubiquitinated bands appeared by 2 min; all diminished after 5 min. To understand if the ubiquitinated Syk was degraded by the proteasomal system, human platelets were pre-treated with proteasomal inhibitors (MG132 or Epoxomicin) followed by activation of FcγRIIA. There was no accumulation of Ub-Syk observed for up to 5 minutes after FcγRIIA stimulation in the presence of proteasomal inhibitors compared to controls, indicating that Ub-Syk is not degraded by the proteasomes and inactivated. Activation of FcγRIIA by cross linking with IV.3/GAM in HEL cells, a cell line model, showed a transient increase in ubiquitylation and phosphorylation of Syk within 15 sec which decreased by 3 min. To understand if activation of Syk by upstream Src-family kinases is necessary for ubiquitylation in this model system, SFK inhibitor (PP2) (10uM) was pre-incubated with HEL cells followed by FcγRIIA activation. Western blot analysis showed elimination of phosphorylated and ubiquitinated Syk upon activation of FcγRIIA in HEL cells in presence of PP2. In contrast, ubiquitylation and phosphorylation of Syk was observed in control cells treated with PP3 or vehicle (DMSO) and as well as in untreated cells. HEL cells were also pretreated with proteasomal inhibitors (MG132 or Epoxomicin) followed by activation of FcγRIIA. There was no accumulation of phosphorylated and ubiquitinated Syk observed for up to 10 min of stimulation of FcγRIIA in HEL cells in presence of proteasomal inhibitors compared to controls indicating no degradation of Ub-syk by proteasomal complex. Finally, we directly compared the ubiquitylation and phosphorylation of Syk in GPVI/FcRγ-stimulated vs.FcγRIIA-stimulated platelets from the same donors. Of note, FcγRIIA-stimulated platelets demonstrated both different kinetics and extent of Syk ubiquitylation than did GPVI/FcRγ-stimulated platelets. In this study, we demonstrated that FcγRIIA signaling results in transient Syk tyrosine phosphorylation and ubiquitylation in platelets and HEL cells. The ubiquitinated Syk is not destined for proteasomal degradation. Limited ubiquitylation of proteins has been noted to modulate downstream signaling events and protein-protein interactions. Further studies are needed to decipher the molecular partners of ubiquitinated Syk following FcγRIIA activation, and to elucidate the differences between GPVI/FcRγ and FcγRIIA signaling via Syk. Disclosures: No relevant conflicts of interest to declare.

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Pamuk, Omer Nuri, Peter H. Lapchak, Poonam Rani, Polly Pine, Jurandir J. Dalle Lucca, and George C. Tsokos. "Spleen tyrosine kinase inhibition prevents tissue damage after ischemia-reperfusion." American Journal of Physiology-Gastrointestinal and Liver Physiology 299, no. 2 (August 2010): G391—G399. http://dx.doi.org/10.1152/ajpgi.00198.2010.

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Reperfusion injury to tissue following an ischemic event occurs as a consequence of an acute inflammatory response that can cause significant morbidity and mortality. Components of both the innate (complement, immunoglobulin, monocytes, and neutrophils) and adaptive (B and T lymphocytes) immune systems have been demonstrated to mediate tissue injury. Spleen tyrosine kinase (Syk) is responsible for membrane-mediated signaling in various cell types including B lymphocytes, macrophages, and T cells. We investigated the ability of a small drug Syk inhibitor, R788, to protect mice against mesenteric ischemia-reperfusion (I/R)-induced local (intestine) and remote lung injury. Mice were fed with chow containing a Syk inhibitor for 6 days before the performance of intestinal I/R, which resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local and remote lung injury. The beneficial effect was associated with reduced IgM and complement 3 deposition in the tissues and significant reduction of polymorphonuclear cell infiltration. Our data place Syk upstream of events leading to the binding of natural antibodies to the ischemia-conditioned tissues and urge the consideration of the use of Syk inhibitors in the prevention or improvement of tissue injury of organs exposed to ischemia or hypoperfusion.

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Biagioli, Michele, Andrea Mencarelli, Adriana Carino, Sabrina Cipriani, Silvia Marchianò, Chiara Fiorucci, Annibale Donini, et al. "Genetic and Pharmacological Dissection of the Role of Spleen Tyrosine Kinase (Syk) in Intestinal Inflammation and Immune Dysfunction in Inflammatory Bowel Diseases." Inflammatory Bowel Diseases 24, no. 1 (December 19, 2017): 123–35. http://dx.doi.org/10.1093/ibd/izx031.

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Abstract Background The DNAX adaptor protein 12 (DAP12) is a transmembrane adaptor molecule that signals through the activation of Syk (Spleen Tyrosine Kinase) in myeloid cells. The purpose of this study is to investigate the role of DAP12 and Syk pathways in inflammatory bowel diseases (IBDs). Methods DAP12 deficient and DAP12 transgenic, overexpressing an increased amount of DAP12, mice and Syk deficient mice in the C57/BL6 background were used for these studies. Colitis was induced by administering mice with dextran sulfate sodium (DSS), in drinking water, or 2,4,6-trinitrobenzene sulfonic acid (TNBS), by intrarectal enema. Results Abundant expression of DAP12 and Syk was detected in colon samples obtained from Crohn’s disease patients with expression restricted to immune cells infiltrating the colonic wall. In rodents development of DSS colitis as measured by assessing severity of wasting diseases, global colitis score,and macroscopic and histology scores was robustly attenuated in DAP12-/- and Syk-/- mice. In contrast, DAP12 overexpression resulted in a striking exacerbation of colon damage caused by DSS. Induction of colon expression of proinflammatory cytokines and chemokines in response to DSS administration was attenuated in DAP12-/- and Syk-/- mice, whereas opposite results were observed in DAP12 transgenic mice. Treating wild-type mice with a DAP-12 inhibitor or a Syk inhibitor caused a robust attenuation of colitis induced by DSS and TNBS. Conclusions DAP12 and Syk are essential mediators in inflammation-driven immune dysfunction in murine colitides. Because DAP12 and Syk expression is upregulated in patients with active disease, present findings suggest a beneficial role for DAP12 and Syk inhibitors in IBD.

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Lauvrak, Silje Ugland, Sébastien Wälchli, Tore-Geir Iversen, Hege Holte Slagsvold, Maria Lyngaas Torgersen, Bjørn Spilsberg, and Kirsten Sandvig. "Shiga Toxin Regulates Its Entry in a Syk-dependent Manner." Molecular Biology of the Cell 17, no. 3 (March 2006): 1096–109. http://dx.doi.org/10.1091/mbc.e05-08-0766.

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Shiga toxin (Stx) is composed of an A-moiety that inhibits protein synthesis after translocation into the cytosol, and a B-moiety that binds to Gb3 at the cell surface and mediates endocytosis of the toxin. After endocytosis, Stx is transported retrogradely to the endoplasmic reticulum, and then the A-fragment enters the cytosol. In this study, we have investigated whether toxin-induced signaling is involved in its entry. Stx was found to activate Syk and induce rapid tyrosine phosphorylation of several proteins, one protein being clathrin heavy chain. Toxin-induced clathrin phosphorylation required Syk activity, and in cells overexpressing Syk, a complex containing clathrin and Syk could be demonstrated. Depletion of Syk by small interfering RNA, expression of a dominant negative Syk mutant (Syk KD), or treatment with the Syk inhibitor piceatannol inhibited not only Stx-induced clathrin phosphorylation but also endocytosis of the toxin. Also, Golgi transport of Stx was inhibited under all these conditions. In conclusion, our data suggest that Stx regulates its entry into target cells.

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Sender, Sina, Ahmad Wael Sultan, Daniel Palmer, Dirk Koczan, Anett Sekora, Julia Beck, Ekkehard Schuetz, et al. "Evaluation of the Synergistic Potential of Simultaneous Pan- or Isoform-Specific BET and SYK Inhibition in B-Cell Lymphoma: An In Vitro Approach." Cancers 14, no. 19 (September 27, 2022): 4691. http://dx.doi.org/10.3390/cancers14194691.

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Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature.

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Hatton, Olivia L., Stacie Lambert, Maria Vaysberg, Sheri M. Krams, Carlos O. Esquivel, and Olivia M. Martinez. "Syk, a novel therapeutic target for PTLD, drives Epstein Barr Virus B cell lymphoma growth and survival through the PI3K/Akt pathway (141.23)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 141.23. http://dx.doi.org/10.4049/jimmunol.182.supp.141.23.

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Abstract Post-transplant lymphoproliferative disorder (PTLD)-associated Epstein Barr Virus (EBV)+ B cell lymphomas are a serious, and often fatal, complication of solid organ and bone marrow transplantation. In EBV+ PTLD, latent membrane protein 2a (LMP2a), a mimic of the B cell receptor, provides a constitutive survival signal to latently infected cells. This signal is transmitted through the sequestering and activation of Lyn and Syk and subsequent activation of downstream survival pathways. We hypothesized that inhibition of Syk activity would diminish growth of EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with R406, a novel Syk inhibitor, decreased proliferation and induced apoptosis of PTLD lines. While the NFkB and p38 pathways were unaffected after PTLD lines were treated with R406, both the Akt and Erk pathways were inhibited. Akt is constitutively active in these cells, and treatment with R406 led to decreased Akt-dependent autocrine IL-10 secretion. These data indicate that Syk mediates its effects on EBV+ PTLD growth and survival through activation of the PI3K/Akt pathway. Syk inhibitors, such as R406, may serve as an effective therapeutic strategy for EBV+ PTLD.

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25

Woulfe, Donna S. "A Syk inhibitor for sick platelets?" Blood 113, no. 14 (April 2, 2009): 3133–34. http://dx.doi.org/10.1182/blood-2009-01-199778.

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Tümmler, Conny, Gianina Dumitriu, Malin Wickström, Peter Coopman, Andrey Valkov, Per Kogner, John Inge Johnsen, Ugo Moens, and Baldur Sveinbjörnsson. "SYK Inhibition Potentiates the Effect of Chemotherapeutic Drugs on Neuroblastoma Cells in Vitro." Cancers 11, no. 2 (February 10, 2019): 202. http://dx.doi.org/10.3390/cancers11020202.

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Neuroblastoma is a malignancy arising from the developing sympathetic nervous system and the most common and deadly cancer of infancy. New therapies are needed to improve the prognosis for high-risk patients and to reduce toxicity and late effects. Spleen tyrosine kinase (SYK) has previously been identified as a promising drug target in various inflammatory diseases and cancers but has so far not been extensively studied as a potential therapeutic target in neuroblastoma. In this study, we observed elevated SYK gene expression in neuroblastoma compared to neural crest and benign neurofibroma. While SYK protein was detected in the majority of examined neuroblastoma tissues it was less frequently observed in neuroblastoma cell lines. Depletion of SYK by siRNA and the use of small molecule SYK inhibitors significantly reduced the cell viability of neuroblastoma cell lines expressing SYK protein. Moreover, SYK inhibition decreased ERK1/2 and Akt phosphorylation. The SYK inhibitor BAY 613606 enhanced the effect of different chemotherapeutic drugs. Transient expression of a constitutive active SYK variant increased the viability of neuroblastoma cells independent of endogenous SYK levels. Collectively, our findings suggest that targeting SYK in combination with conventional chemotherapy should be further evaluated as a treatment option in neuroblastoma.

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27

Hoellenriegel, Julia, Greg Coffey, Uma Sinha, Anjali Pandey, William G. Wierda, Michael J. Keating, and Jan A. Burger. "Spleen Tyrosine Kinase (Syk) Inhibitors Block B Cell Receptor Signaling and Survival In Chronic Lymphocytic Leukemia." Blood 116, no. 21 (November 19, 2010): 3604. http://dx.doi.org/10.1182/blood.v116.21.3604.3604.

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Abstract Abstract 3604 B cell antigen receptor (BCR) signaling is increasingly recognized as a key factor promoting clonal expansion in various B cell malignancies, such as diffuse large B cell lymphoma and CLL. Engagement of the BCR receptor activates Syk, which leads to a number of downstream events that promote cell survival and growth. Therefore, inhibition of Syk represents a novel therapeutic approach in CLL. Another Syk inhibitor (fostamatinib disodium/R788) has clinical activity in patients with CLL (Blood 115: 2570, 2010). R788 is a relatively selective Syk inhibitor, but also displays activity against Flt3, Jak, and Lck. In this study we tested two highly selective Syk inhibitors (P142-76 and P505-15) and a multi-kinase inhibitor (P420-89) for their efficacy to antagonize BCR-related CLL cell activation and survival responses. We found that BCR crosslinking with anti-IgM significantly increased CLL cell viability compared to controls, and this pro-survival effect of BCR triggering was abrogated by treatment with the Syk inhibitors P142-76 and P505-15. Figure A shows contour plots that depict CLL cell viability in a representative case, treated with anti-IgM in the presence or absence of the Syk inhibitors. The mean relative CLL cell viability at 48 hours was decreased to 78% ± 4% (P142-76), 62% ± 5% (P505-15) or 50% ± 4.5% (P420-89) of controls (means ± SEM, n=19). Additionally, we found that the inhibitors induce apoptosis in CLL cells in co-culture with nurselike cells (NLC), indicating that Syk inhibition antagonizes microenvironment-derived survival signals which may or may not be related to the BCR. For example, P505-15 significantly reduced CLL cell viability in NLC co-cultures from 84.4% ± 5% to 46% ± 8% at 48 hours (mean ± SEM, n=6,*P< 0.05, summarized in Fig. B). The chemokines CXCL12 and CXCL13 regulate migration and homing of CLL cells within the CLL microenvironment, and CLL cell responsiveness to these chemokines is modulated by BCR signaling. Therefore, we evaluated the effects of the Syk inhibitors on CLL cell chemotaxis towards these chemokines. P505-15 decreased chemotaxis toward CXCL12 and CXCL13 to levels that were 49.5% ± 5% or 32.8% ± 6% of respective controls. In response to BCR crosslinking, CLL cells secrete the chemokines CCL3 and CCL4, which foster interactions between CLL cells and the leukemia microenvironment. This was almost completely abrogated by inhibiting Syk. For example, preincubation with P505-15 significantly reduced CCL3 supernatant levels from 8400 pg/mL ± 1166 pg/mL to 2263 pg/mL ± 744 pg/mL (mean ± SEM, n=5, *P< 0.05). This inhibition of CCL3/4 secretion by CLL cells could also be demonstrated in CLL co-cultures with NLC. Finally, we found that P505-15 blocked BCR-induced activation of the p44/42 MAP kinase, using anti-phospho-MAPK (ERK1/2) antibodies. In summary, our findings demonstrate that specific Syk inhibitors are highly effective in disrupting BCR-derived survival and cell migration-related responses in CLL cells. These data support the clinical development of these new, promising agents in patients with CLL and other selected B cell malignancies. Disclosures: Coffey: Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment.

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Pasquet, Jean-Max, Romain Gioia, Claire Drullion, Valerie Lagarde, Cedric Leroy, Serge Roche, Bruno Cardinaud, and Francois-Xavier Mahon. "Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass." Blood 114, no. 22 (November 20, 2009): 2175. http://dx.doi.org/10.1182/blood.v114.22.2175.2175.

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Abstract Abstract 2175 Poster Board II-152 Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl. Disclosures: Mahon: Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

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Zheng, Tony, Elizabeth R. Lofurno, Sarah Elgamal, Alexander R. Melrose, Jiaqing Pang, Joseph J. Shatzel, Owen J. T. McCarty, and Joseph Aslan. "Tyrosine Kinase Inhibitors (TKIs) Targeting Syk and BTK Signaling Differentially Affect PI3K Signalosome Organization and Platelet Function." Blood 134, Supplement_1 (November 13, 2019): 2074. http://dx.doi.org/10.1182/blood-2019-129066.

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Small molecule tyrosine kinase inhibitors (TKIs) serve increasingly important roles in the treatment of hematologic malignancies and also have shown efficacy in a range of immunological and inflammatory conditions. However, many successful TKI therapies are associated with problematic effects - particularly on platelets - as bleeding complications have been reported for many TKIs, both on and off clinical trials. Bleeding risk is especially apparent for patients treated with ibrutinib and other irreversible inhibitors of Bruton's tyrosine kinase (BTK), several of which now effectively treat multiple B cell malignancies. Intriguingly, other therapeutic TKIs targeting mechanistic partners of BTK are not associated with platelet-related complications. For instance, inhibitors against the BTK-activating kinase Syk - both with selective (i.e., entospletinib) as well as promiscuous (i.e., fostamatinib) target profiles - are not associated with clinically increased bleeding, despite some reported antiplatelet effects. To gain insight into the multifaceted effects of Syk and BTK inhibitors on platelet function, we analyzed the effects of a panel of eight different clinically relevant Syk- and BTK-directed TKIs (i.e., fostamatinib/R406, entospletinib, ibrutinib, acalabrutinib, AVL-292 and others) on essential platelet responses. We first assessed the effects of TKI treatment on Syk and BTK-mediated signaling events downstream of the platelet immunoreceptor tyrosine-based activation motif (ITAM) receptor GPVI. All Syk and BTK TKIs tested inhibited the phosphorylation of phospholipase C (PLCγ2) Y1217, protein kinase Cδ (PKCδ) Y311 and Akt T308, as well as Akt substrate phosphorylation following platelet stimulation with the GPVI receptor agonist, crosslinked collagen-related peptide (CRP-XL). Independent of TKI target profile, Syk and BTK TKIs differentially inhibited the phosphorylation of DAPP1 Y132, phosphoinositide 3-kinase (PI3K) p85α Y458, Akt S473 and PKC substrates. Syk and BTK TKIs also inhibited platelet adhesion to immobilized CRP-XL in a manner matching the effects of each TKI on ITAM-mediated signaling events. However, platelet signaling and adhesion phenotypes did not match the effects of these same TKIs on GPVI-triggered dense granule secretion, which was inhibited by all TKIs tested with the exception of R406. In contrast, all Syk and BTK inhibitors tested inhibited platelet spreading on immobilized fibrinogen. Based on observations above, we hypothesized that TKIs that irreversibly bind to BTK may disrupt critical molecular interactions around BTK within the PI3K signalosome essential to the proper orchestration of platelet activation programs. To test the effects of Syk and BTK inhibitors on the organization of PI3K signaling in activating platelets, platelets were pretreated with TKIs prior to incubation on immobilized fibrinogen and processing for immunofluorescence microscopy. We found that under control conditions, PI3K p85α regulatory subunit localized to phospholipid PI(3,4)P2-rich regions in adherent platelets associated with active PKC signaling. While Syk and PI3K inhibitors abrogated platelet spreading on fibrinogen, PI3K p85α staining remained strong around undeveloped, nascent adhesions in platelets treated with Syk and PI3K inhibitors. In contrast, PI3K p85α staining was diffuse, diminished or absent in platelets adherent to fibrinogen in the presence of all irreversible BTK inhibitors tested. Our results show that clinically effective kinase inhibitors that target Syk-BTK-PI3K signaling systems have varying effects on platelet function that may offer insights to adverse effects of therapeutic TKIs in different contexts. Moreover, our findings suggest that in addition inhibiting protein kinase activities, the discrepant effects of some TKIs may be attributed to, in part, altered protein relations around BTK, PLCγ2 and other components of the PI3K signalosome in activating platelets. Ongoing studies aim to specify protein:protein interactions affected by reversible as well as irreversible TKIs; to examine patient samples for evidence of PI3K mislocalization - especially in cases of BTK inhibitor-associated bleeding; and, to determine if PI3K signalosome disorganization has roles in other physiological, therapeutic and toxic effects of TKIs. Disclosures Shatzel: Aronora, Inc.: Consultancy.

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Hahn, Cynthia K., Kenneth N. Ross, Rose M. Kakoza, Steven A. Carr, Jinyan Du, Shao-En Ong, Todd R. Golub, and Kimberly Stegmaier. "Syk Is a New Target for AML Differentiation." Blood 110, no. 11 (November 16, 2007): 209. http://dx.doi.org/10.1182/blood.v110.11.209.209.

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Abstract One of the challenges of phenotype-based small molecule screening has been the difficulty of protein target identification for newly discovered compounds. For example, we previously performed a gene expression-based small molecule library screen, which identified gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, as an inducer of acute myeloid leukemia (AML) differentiation in cell lines and primary patient cells. Neither EGFR transcript nor protein is expressed in the tested AML cell lines, thus precluding inhibition of this kinase as the mechanism of AML differentiation. However, because multiple EGFR inhibitors induce this phenotype, we hypothesize that a shared off-target kinase is the target in AML differentiation. In order to identify candidate gefitinib targets of AML differentiation, we utilized a proteomics method: peptide immunoprecipitation-HPLC-mass spectrometry. This approach enriches for phospho-tyrosine peptides with immunoprecipitation (IP) after enzyme digestion. In contrast to IP at the protein level, the final mixture following peptide-IP contains primarily phospho-tyrosine-containing peptides. We treated the AML cell line HL-60 with gefitinib versus vehicle and then identified peptide sites with loss of phosphorylation with gefitinib treatment. Spleen tyrosine kinase (Syk) was identified as one of the only kinases with loss of phosphorylation post treatment. Syk is a nonreceptor tyrosine kinase, important in normal B-cell differentiation, and implicated in malignancies such as myelodysplastic syndrome and lymphoma. We first confirmed with IP western immunoblotting the inhibition of Syk phosphorylation with gefitinib treatment in HL-60 cells. We next confirmed with both pharmacological inhibition and genetic loss of Syk the induction of differentiation in the AML cell lines HL-60 and U937. Numerous determinants of myeloid maturation were tested: a complex differentiation gene expression signature, cellular morphology, cell surface proteins CD11b and CD14 expression, and nitro-blue tetrazolium (NBT) reduction, a functional assay for myeloid differentiation. Two reported pharmacological inhibitors of Syk scored positive on all measurements of differentiation. Furthermore, the shRNA construct inducing the most complete loss of Syk also scored the highest on all measurements of differentiation. The most convincing data that Syk is the true target of these inhibitors would be the identification of an inhibitor resistant Syk mutant, analogous to BCR-ABL or c-Kit mutants that confer imatinib resistance. To this end, a Syk random mutagenesis screen is ongoing. In summary, these data identify Syk as a strong candidate target of gefitinib, demonstrate that inhibition of Syk can induce AML differentiation, and identify Syk as a potential target for AML differentiation therapy.

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Lin, Yipeng. "Kinase inhibitor therapies for Chronic lymphocytic leukaemia (CLL): SYK, BTK and PI3K inhibitors." Highlights in Science, Engineering and Technology 19 (November 17, 2022): 30–35. http://dx.doi.org/10.54097/hset.v19i.2691.

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Chronic lymphocytic leukaemia (CLL) is a prevalent tumor disease in developed countries, and related therapies have been designed. However, CLL is still incurable. Chemoimmunotherapy is effective in inhibiting the proliferation of CLL cells, but nonspecific treatment can affect the growth of other immune cells. Kinase inhibitors are considered to be effective treatments for CLL as their anti-proliferation effects, and currently, popular kinase inhibitor therapies include SYK, BTK, and PI3K inhibitor therapy. PI3K is characterized by high efficiency and low side effects compared with the other two kinase inhibitor therapies, for instance, idelalisib and duvelisib. This review compares the advantages of each kinase inhibitor therapy through relevant studies and concludes that duvelisib has significant advantages and promising prospects compared to other CLL drugs. Further research may focus on exploring the mechanism of the role of kinase inhibitors in CLL as well as the clinical trials of kinase inhibitors in CLL patients.

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YAMAOKA, Kunihiro, Kazuyoshi SAITO, and Yoshiya TANAKA. "4. Drugs Targeting Intracellular Molecule: JAK Inhibitor and Syk Inhibitor." Rinsho yakuri/Japanese Journal of Clinical Pharmacology and Therapeutics 44, no. 1 (2013): 23–27. http://dx.doi.org/10.3999/jscpt.44.23.

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Perrella, Gina, Samantha J. Montague, Helena C. Brown, Lourdes Garcia Quintanilla, Alexandre Slater, David Stegner, Mark Thomas, Johan W. M. Heemskerk, and Steve P. Watson. "Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear." International Journal of Molecular Sciences 23, no. 1 (January 1, 2022): 493. http://dx.doi.org/10.3390/ijms23010493.

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Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s−1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

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Rezaul, Karim, Shigeru Yanagi, Kiyonao Sada, Takanobu Taniguchi, and Hirohei Yamamura. "Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets." Thrombosis and Haemostasis 72, no. 06 (1994): 937–41. http://dx.doi.org/10.1055/s-0038-1648987.

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SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

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Balaian, Larissa, and Edward D. Ball. "The Anti-Proliferative Effect of Immunotoxin Gemtuzimab Ozogamycin (GO) (Mylotarg) to Acute Myeloid Leukemia (AML) Cells Is Associated with the Level of Protein Kinase Syk Exspression." Blood 104, no. 11 (November 16, 2004): 96. http://dx.doi.org/10.1182/blood.v104.11.96.96.

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Abstract AML cells express the cell surface antigen CD33 that serves as a down-regulator of cell growth. Anti-CD33 monoclonal antibody (mAb) coupled to a toxin is a licensed drug for the treatment of relapsed AML (Mylotarg). Syk is not only an essential element in several cascades coupling antigen receptors to cell responses, but also SYK is a tumor suppressor gene. Silencing of SYK by hypermethylation results in absence of Syk expression and unresponsiveness of tumor cells to various treatments.. Earlier we demonstrated that about 30% of the AML samples were Syk-negative and the response of leukemia cells to CD33 ligation correlated with Syk expression. Therefore, here we investigated whether or not the response of the AML cells to GO treatment also depends on the level of Syk expression. It is even more intriguing since 50% of Mylotarg is not conjugated to calicheamicin, and hence some of its activity is in fact due to anti-CD33 mAb signaling. Primary leukemia cells were analyzed for their level of the Syk expression (11 Syk positive and 6 Syk-negative) and then treated with GO. In both groups GO mediated dose-dependent inhibition of proliferation, however the level of inhibition in Syk-positive group was significantly higher (p<0.003). The largest differences were notices at low doses (1–50 ng/ml) of GO, while at higher doses (>100 ng/ml) this effect was diminished. Moreover, 8 of 11 (72.7%) samples in Syk-positive group were “good” responders to GO (inhibition>50%), while in Syk-negative group only 1 of 6 (16.6%) samples demonstrated similar response. This difference was statistically significant and suggesting a correlation between the level of Syk expression in AML cells and their response to Mylotarg. After treatment of Syk-negative AML samples with methylation inhibitor 5-aza-cytidine in 2 of 6 samples Syk protein expression was restored. However, in all 6 samples after 5-aza-CdR treatment GO inhibitory activity was significantly higher (p<0.005). Moreover, after 5-aza-cytidine treatment, these initially CD33 ligation non-responsive cells demonstrated high level (up to 70%) inhibition of proliferation upon anti-CD33 mAb treatment. Analysis of CD33 ligation mediated signaling in 3 Syk-positive samples (1 responsive and 2 unresponsive to GO treatment) revealed significant differences: in responsive AML sample Syk became phosphorylated and created complexes with CD33 and protein phosphatase SHP-1, while in non-responsive samples were no Syk/CD33 or Syk/SHP-1 complexes. This does imply that not only the physical presence of Syk, but also its functional activity is important for the adequate response of AML cells to Mylotarg. To create a model with down-regulated Syk we treated Syk-positive human AML cell lines with various doses of the Syk inhibitor Piceatannol and analyzed their proliferative response to Mylotarg. In AML cell lines treated with 10–100 μM of Piceatannol, GO inhibitory activity was significantly lower compared to untreated cells or even those treated by low doses of Piceatannol. This confirmed our observation that expression and functional activity of Syk is associated with the ability of AML cells to respond to GO treatment. We hypothesize that the pre-treatment level of Syk expression may be used as a prognostic factor for response to Mylotarg treatment. Screening for Syk expression in leukemia cell samples from patients before treatment might allow rational selection of patients for this expensive and toxic therapy and thus improve its success rate in the clinic.

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Buchner, Maike, Constance Baer, Gabriele Prinz, Christine Dierks, Meike Burger, Thorsten Zenz, Stephan Stilgenbauer, Hassan Jumaa, Hendrik Veelken, and Katja Zirlik. "Spleen tyrosine kinase inhibition prevents chemokine- and integrin-mediated stromal protective effects in chronic lymphocytic leukemia." Blood 115, no. 22 (June 3, 2010): 4497–506. http://dx.doi.org/10.1182/blood-2009-07-233692.

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Abstract The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.

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Gaino, Stefania, Valeria Zuliani, Rosa Tommasoli, Donatella Benati, Riccardo Ortolani, Carlo Zancanaro, Giorgio Berton, Clara Santonastaso та Pietro Minuz. "Functional Role of p38 Mitogen Activated Protein Kinase in Platelet Activation induced by a Thromboxane A2 Analogue and by 8-iso-prostaglandin F2 α". Thrombosis and Haemostasis 87, № 05 (2002): 888–98. http://dx.doi.org/10.1055/s-0037-1613101.

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SummaryWe investigated similarities in the signaling pathways elicited by the F2 isoprostane 8-iso-PGF2α and by low doses of U46619 to induce platelet activation. Both 0.01-0.1 µmol/L U46619 and 0.01-1 µmol/L 8-isoPGF2α triggered shape change and filopodia extension, as well as adhesion to immobilized fibrinogen of washed platelets. At these doses the two platelet agonists failed to trigger secretion and aggregation, which were however induced by higher doses of U46619 (0.1-1 µmol/L). SB203580 (1-10 µmol/L), a specific inhibitor of the p38 mitogen activated protein (MAP) kinase blunted platelet shape change and adhesion induced by 0.05-1 µmol/L 8-iso-PGF2α and by 0.01 µmol/L U46619. These platelet responses were also inhibited by 20 µmol/L cytochalasin D, an inhibitor of actin polymerization, and 50 µmol/L piceatannol, an inhibitor of the Syk tyrosine kinases. Both 8-iso-PGF2α and U46619-induced p38 MAP kinase phosphorylation in suspended platelets and this was inhibited by piceatannol, indicating that Syk activation occurs upstream p38 MAP kinase phosphorylation. These findings suggest that the signaling pathway triggered by both 8-iso-PGF2α and low concentrations of U46619 to induce platelet adhesion and shape change implicates Syk, the p38 MAP kinase, and actin polymerization.

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Fernandez, Rosemarie, and Suzanne J. Suchard. "Syk Activation Is Required for Spreading and H2O2 Release in Adherent Human Neutrophils." Journal of Immunology 160, no. 10 (May 15, 1998): 5154–62. http://dx.doi.org/10.4049/jimmunol.160.10.5154.

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Abstract Chemoattractant-stimulated polymorphonuclear leukocytes (PMNs) that are adherent to extracellular matrix proteins exhibit a massive, sustained respiratory burst that requires cell spreading. However, the signaling pathways culminating in PMN spreading are not well characterized. Studies showing that protein tyrosine phosphorylation increases with PMN spreading suggest that phosphorylation is critical for this process. In the present study, we observed increased tyrosine phosphorylation of both focal adhesion kinase and Syk in FMLP-activated PMNs that had been plated onto fibrinogen; an increase in Syk activity, but not focal adhesion kinase activity, was apparent. The time course of Syk phosphorylation correlated with the initiation of cell spreading and H2O2 release. Pretreatment of PMNs with piceatannol, a Syk-selective inhibitor, blocked Syk activity, cell spreading, and H2O2 release, indicating that Syk activity was required for the activation of adherent PMNs. Paxillin is a cytoskeletally associated protein that is also tyrosine phosphorylated during PMN spreading and H2O2 release. Paxillin phosphorylation is kinetically slower than Syk phosphorylation and is inhibited with piceatannol, suggesting that paxillin is a substrate for Syk. An analysis of Syk immunoprecipitates indicated that Syk and paxillin associate during PMN spreading. This interaction is not mediated by the src kinases Lyn and Fgr, since neither kinase coprecipitated with Syk. Syk from FMLP-activated, adherent PMNs phosphorylated paxillin-glutathione S-transferase, suggesting that paxillin is a substrate for Syk in vivo. These results indicate that PMN spreading and H2O2 release require a Syk-dependent signaling pathway leading to paxillin phosphorylation.

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39

Law, Debbie A., Lisa Nannizzi-Alaimo, Kathleen Ministri, Paul E. Hughes, Jane Forsyth, Martin Turner, Sanford J. Shattil, Mark H. Ginsberg, Victor L. J. Tybulewicz та David R. Phillips. "Genetic and Pharmacological Analyses of Syk Function in IIbβ3 Signaling in Platelets". Blood 93, № 8 (15 квітня 1999): 2645–52. http://dx.doi.org/10.1182/blood.v93.8.2645.

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Abstract Agonists induce inside-out IIbβ3signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in IIbβ3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of IIbβ3 by adenine diphosphate (ADP) ± epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in IIbβ3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits IIbβ3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.

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40

Law, Debbie A., Lisa Nannizzi-Alaimo, Kathleen Ministri, Paul E. Hughes, Jane Forsyth, Martin Turner, Sanford J. Shattil, Mark H. Ginsberg, Victor L. J. Tybulewicz та David R. Phillips. "Genetic and Pharmacological Analyses of Syk Function in IIbβ3 Signaling in Platelets". Blood 93, № 8 (15 квітня 1999): 2645–52. http://dx.doi.org/10.1182/blood.v93.8.2645.408k13_2645_2652.

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Agonists induce inside-out IIbβ3signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in IIbβ3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of IIbβ3 by adenine diphosphate (ADP) ± epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in IIbβ3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits IIbβ3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.

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41

Mullard, Asher. "FDA approves first-in-class SYK inhibitor." Nature Reviews Drug Discovery 17, no. 6 (June 2018): 385. http://dx.doi.org/10.1038/nrd.2018.96.

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42

Patou, Joke, Gabriele Holtappels, Karen Affleck, Paul van Cauwenberge, and Claus Bachert. "Syk-kinase inhibition prevents mast cell activation in nasal polyps." Rhinology journal 49, no. 1 (March 1, 2011): 100–106. http://dx.doi.org/10.4193/rhino09.147.

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Background: Mast cells are crucial effector cells in the allergic cascade. The cross-linking of the high affinity IgE receptor (FcεRI) activates mast cells and basophils. Spleen tyrosine kinase (Syk) is positioned upstream of the IgE receptor signal transducing pathway and may represent an important target for the treatment of nasal inflammatory diseases. Objective: We measured effects of a specific Syk inhibitor in the release of mast cell mediators in human cord blood-derived mast cells (CBDMCs) (in-vitro) and in human nasal tissue (ex-vivo). Methods: Surgical samples were collected from patients with nasal polyposis who underwent sinus surgery. Tissue cubes of +- 0.9 mm3 were primed with myeloma IgE (1 microg/ml), preincubated with Syk inhibitor NVP-QAB205 in different concentrations and then stimulated with tissue culture medium, anti-IgE 10 microg/ml and anti-IgE 30 microg/ml. Supernatants were analysed for concentrations of histamine, LTC4/LTD4/LTE4 and PGD2. CBDMCs were likewise pre-incubated with compound, prior to stimulation with anti-IgE at 10 microg/ml. Results: In CBDMCs, the Syk inhibitor prevented degranulation assessed by measurement of histamine release and the production of LTC4/LTD4/LTE4 and PGD2. Furthermore, the Syk inhibitor was similarly able to significantly inhibit the release of these granules and newly synthesized mediators by nasal polyp mast cells in a dose dependent manner. Conclusion: Although the critical role of Syk in the IgE receptor signal transduction pathway has been well documented in vitro, this study supports the importance of Syk in IgE receptor-mediated degranulation of mast cells ex-vivo within nasal tissue. Thus, inhibition of Syk may represent an important therapeutic strategy for the treatment of upper airway disease with mast cell involvement, such as allergic rhinitis.

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43

Cheng, Shuhua, Greg Coffey, X. Hannah Zhang, Rita Shaknovich, Zibo Song, Pin Lu, Anjali Pandey, Ari M. Melnick, Uma Sinha, and Y. Lynn Wang. "SYK inhibition and response prediction in diffuse large B-cell lymphoma." Blood 118, no. 24 (December 8, 2011): 6342–52. http://dx.doi.org/10.1182/blood-2011-02-333773.

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Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G1-S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLCγ2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLCγ2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors.

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44

Kinouchi, Chieko, Kazuya Taguchi, Susumu Shimoyama, Tadaaki Ioroi, Hayato Ogura, Mari Yamamoto, Akiko Iino, et al. "FF-10102-01: A Novel, Highly Selective Spleen Tyrosine Kinase Inhibitor, to Be in Clinical Application for Treatment of Autoimmune Diseases and B-Cell Malignancies." Blood 128, no. 22 (December 2, 2016): 3745. http://dx.doi.org/10.1182/blood.v128.22.3745.3745.

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Abstract Spleen tyrosine kinase (Syk) is essential for downstream pathways in signaling from B-cell receptor (BCR) in B cells and Fc-gamma receptors in macrophages. Because of the essential roles it plays in signaling, Syk has been targeted in drug development for autoimmune and allergic diseases, in which B cells and macrophages have pivotal roles in the pathophysiology. In addition, accumulated data suggest that aberrant BCR signaling is deeply involved in the pathogenesis of B-cell malignancies. Here we report the pharmacological profile of a novel and highly selective Syk inhibitor, FF-10102-01 (FF-10102 hydrochloride). FF-10102-01 was evaluated in the following in vitro assays: Kinase inhibitory activities were performed with ProfilerPro Kit (PerkinElmer, USA). The effects on cell signaling and functions were studied using THP-1, a human monocyte-derived cell line, after differentiation with human recombinant interferon gamma (IFNγ). The effects on CD69, an activated B-cell marker, expression on B cells were evaluated using whole blood samples from healthy volunteers and mice, and on lymphoma cell growth using SU-DHL-6, a human diffuse large B-cell lymphoma-derived cell line. FF-10102-01 was also evaluated in vivo in animal models of antigen-induced antibody production, anti-platelet antibody-induced thrombocytopenia, and collagen-induced arthritis (CIA). FF-10102-01 showed high potency against recombinant human Syk enzyme activity, with a 50% inhibitory concentration (IC50) value of 1.2 nmol/L. Kinase profiling assay in a panel of 216 kinases revealed that FF-10102-01 is highly selective for Syk, with an IC50 value more than 25 times lower than those of the other kinases. FF-10102-01 exhibited suppressive effects on phosphorylation of downstream molecules of Syk, SLP76, and ERK1/2 in THP-1 cells. FF-10102-01 inhibited tumor necrosis factor α secretion under stimulation by immunoglobulin (Ig) G and phagocytosis of IgG-opsonized beads in IFNγ-induced differentiated THP-1 cells. CD69 expression under stimulation with anti-BCR antibodies in human and mouse blood was also inhibited by FF-10102-01, with IC50 values of 314 nmol/L and 307 nmol/L, respectively. FF-10102-01 was the most potent inhibitor of the growth of SU-DHL-6 cells among other known Syk, JAK, and Bruton's tyrosine kinase inhibitors compared, with an IC50 value of 318 nmol/L. FF-10102-01 is orally available with a good PK profile, and showed inhibitory effect at daily doses 50 mg/kg (as free base) on specific antibody production in ovalbumin-immunized mouse model. Orally administered FF-10102-01 showed significant effects in a mouse model of thrombocytopenia and a rat model of rheumatoid arthritis (CIA) at 25 mg/kg and 10 mg/kg (as free base), respectively. In the thrombocytopenia model, the prevention of platelet decrease by FF-10102-01 was well correlated with suppressions of platelet-phagocytotic macrophages in spleen and CD69-positive B cells in blood. These data indicate that FF-10102-01 is a potent and highly selective orally available Syk inhibitor, with pharmacological effects through inhibition of both B-cell and macrophage activities represented in inhibitions of cell signaling and functions in vitro, and in animal models of autoimmune diseases in vivo. The results suggest that FF-10102-01 is a promising agent for treatment of autoimmune diseases such as immune thrombocytopenia, rheumatoid arthritis, and B-cell malignancies. A clinical trial of FF-10102-01 is planned to commence in 2017. Disclosures Kinouchi: FUJIFILM Corporation: Employment. Taguchi:FUJIFILM Corporation: Employment. Shimoyama:FUJIFILM Corporation: Employment. Ioroi:FUJIFILM Corporation: Employment. Ogura:FUJIFILM Corporation: Employment. Yamamoto:Toyama Chemical Co., Ltd.: Employment. Iino:Toyama Chemical Co., Ltd.: Employment. Maeda:Toyama Chemical Co., Ltd.: Employment. Kato:Toyama Chemical Co., Ltd.: Employment. Fujiwara:FUJIFILM Corporation: Employment. Hagiwara:FUJIFILM Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Kuter:Rigel: Consultancy, Research Funding; Genzyme: Consultancy; Bristol-Myers Squibb: Research Funding; GlaxoSmithKline: Consultancy; ONO: Consultancy; Amgen: Consultancy, Paid expert testimony; Protalex: Research Funding; MedImmune: Consultancy; Pfizer: Consultancy; Syntimmune: Consultancy; Shire: Consultancy; Eisai: Consultancy; 3SBios: Consultancy; CRICO: Other: Paid expert testimony; Shionogi: Consultancy. Nakamura:Toyama Chemical Co., Ltd.: Employment. Shimada:FUJIFILM Corporation: Employment.

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45

Yang, Won Seok, Joon-Seok Kim, Nam Jeong Han, Mee Jeong Lee, and Su-Kil Park. "Toll-Like Receptor 4/Spleen Tyrosine Kinase Complex in High Glucose Signal Transduction of Proximal Tubular Epithelial Cells." Cellular Physiology and Biochemistry 35, no. 6 (2015): 2309–19. http://dx.doi.org/10.1159/000374034.

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Background/Aims: High glucose activates spleen tyrosine kinase (Syk) in human proximal tubular epithelial cells (HK-2 cells), which leads to NF-κB activation and transforming growth factor-ß1 (TGF-ß1) production. We explored the signal transduction pathway from high glucose to Syk activation. Methods: The pathway was evaluated by siRNA transfection, immunoprecipitation and Western blot. Results: High glucose stimulated Syk activation within 10 min. Depletion of toll-like receptor 4 (TLR4) attenuated high glucose-induced Syk activation, NF-κB p65 nuclear translocation, and TGF-ß1 production. In addition, TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody, and depletion of myeloid differentiation factor 88 (MyD88) all attenuated high glucose-induced Syk activation. As an evidence of TLR4 activation, interleukin-1 receptor-associated kinase 1 was recruited to MyD88 and TLR4 upon exposure to high glucose. Syk was co-immunoprecipitated with TLR4, and Syk bound to TLR4 was activated by high glucose. High-mobility group box-1 (HMGB-1), an endogenous activator of TLR4, rapidly increased in TLR4 immunoprecipitates upon high glucose stimulation, and this association was reduced by N-acetylcysteine, an antioxidant. An HMGB-1 inhibitor glycyrrhizin suppressed high glucose-induced Syk activation. Conclusion: Syk is constitutively associated with TLR4. High glucose induces an immediate, reactive oxygen species-dependent, extracellular release of HMGB-1 which binds to TLR4 and activates it, leading to Syk activation.

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46

Kuroda, Etsushi, Keiichi Ohata, Cevayir Coban, and Ken Ishii. "Particulates induce macrophages to produce lipid mediators via the Syk activation. (172.25)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 172.25. http://dx.doi.org/10.4049/jimmunol.188.supp.172.25.

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Abstract Some particulate such as silica crystal (silica), aluminum salts (alum) and monosodium urate (MSU) can stimulate the innate immune system to induce inflammatory responses. Recently, we found that silica and alum induce macrophages to produce both IL-1β and lipid mediator prostaglandin E2 (PGE2). We also indicated that particulate-induced PGE2 is involved in the production of IgE in vivo. However, the underlying mechanisms involved in the induction of PGE2 by particulate remain to be elucidated. In this study, we show that Syk plays an important role in particulate-induced PGE2 production. Silica and alum induced macrophages to produce IL-1β and PGE2. However, NALP3 inflammasome-deficient macrophages produced higher amounts of PGE2 comparable to WT macrophages. To determine which signaling pathway is involved in the production of PGE2, we analyzed PGE2 production from macrophages treated with various signaling inhibitors. Interestingly, we found a Syk inhibitor markedly suppressed PGE2 production. Similar to inhibitor experiment, knockdown of Syk by siRNA in macrophages significantly reduced both PGE2 and IL-1β production. In conclusion, we have found that particulate silica, alum and MSU stimulate macrophages to produce lipid mediator PGE2 though a pathway that is dependent on Syk but independent of the NALP3 inflammasome.

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47

Hartman, Amy D., Annique Wilson-Weekes, Theresa Ruffin, Katie J. Hincher, Larry D. Cripe, and H. Scott Boswell. "Mechanistic Paradigm for Combination Signal Transduction Inhibitor Therapy in Zap70+ CLL Involves Bicompartmental Functions of EZH2." Blood 110, no. 11 (November 16, 2007): 3099. http://dx.doi.org/10.1182/blood.v110.11.3099.3099.

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Abstract We have characterized a tonically active B cell receptor signaling module of zap70+ve CLL cells that contributes to a high-risk disease phenotype associated with rapid tumor doubling time and cellular bcl-2 overexpression. In CLL cells, syk directs tyrosine phosphorylation of certain adaptors including cbl and crkL and this is also accompanied by tyrosine phosphorylation of the interdomain-B loop of zap70 to affect its own interaction with adaptors and vav1. Vav1 along with zap70 allows JNK activation and the formation of heightened c-jun/AP-1 involved in CLL cell bcl-2 transcription. However, CLL pathogenesis is equally dependent upon ezh2/PRC2-mediated 5′-methylation of CpG islands in several tumor suppressor genes such as PTPRO (a cell-intrinsic syk inhibitor) and DAPK1. In addition, ezh2 functions not only in the nucleus for gene repression, but in cytoplasm for stabilization/perpetuation of a zap70-vav1 signal complex upstream of JNK1 in lymphocytes (IH Su et al., Cell121:425, 2005). We investigated the possible participation of ezh2 in events relevant to the outcome of syk inhibition as a primary therapy for CLL. We used R406/R788, a potent, selective syk inhibitor now in clinical trial (Rigel Pharmaceuticals). We found that R406 treatment strongly inhibited CLL viable cell recovery from culture, and this was associated with inhibition of syk (PY525/catalytic domain) phosphorylation as well as loss of zap70 PY319 (interdomain) phosphorylation with an IC50 of 1 uM R406, associated with downregulation of phospho-c-jun (S73) and of bcl-2. Failure of R406 to inhibit lyn autophosphorylation suggests strongly that syk is the upstream kinase for zap70 in this regulatory pathway. A survey of ezh2 expression showed content of ezh2 correlated with p(319)-zap70 intensity in different CLL cells. R406 caused release of syk substrate cbl from the JNK1 signalosome of R406-treated cells, but ezh2 remained tightly bound to vav1-zap70-JNK1 as demonstrated by immunoprecipitation. To overcome the bi-compartmental functions of ezh2, we used HDAC inhibitor, SBHA (suberoyl hydroxamic acid); a class of compound reported to deplete ezh2 from leukemia cells (W Fiskus et al. Mol Canc. Ther.5: 3096, 2006). In 3 different experiments, SBHA alone, 1 uM, had little inhibitory activity for CLL cell viable cell recovery, however, when combined with R406 (also at 1uM) SBHA caused massive cell death (>80%) only attainable with R406 alone at 2.5X dose. Mechanistic aspects of the reversal of tumor suppressor gene repression and of inactivation of the JNK1 signalosome by SBHA are under scrutiny to fully explain its cooperativity with R406, focused especially on the duality of roles performed by ezh2, and the depletion of ezh2 affected by HDAC inhibition.

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48

Körber, Ruth-Miriam, Stefanie AE Held, Solveig Daecke, Marie von Lilienfeld-Toal, Anita Bringmann, and Peter Brossart. "Targeting Syk (spleen tyrosine kinase) Inhibits the Proliferation, Migration and Viability of Multiple Myeloma Cells." Blood 116, no. 21 (November 19, 2010): 4071. http://dx.doi.org/10.1182/blood.v116.21.4071.4071.

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Abstract Abstract 4071 Introduction: Multiple myeloma (MM) is a malignant plasma cell disorder characterized by clonal expansion of single plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of active drugs like bortezomib or lenalidomide that have been associated with improved survival MM is still incurable. Thus, the identification of novel molecular targets and additional treatment options are warranted. In B-cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma, the inhibition of the tyrosine kinase Syk gave promising preclinical and first clinical results.In our study, we analyzed the potential of Syk as a target in MM. Methods: The MM cell lines AMO-1, U266, MMS-1 and RPMI8226 and primary MM cells were treated with the Syk-inhibitors BAY61-3606 or piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Results: Incubation of MM cell lines with Syk-inhibitors resulted in a reduced proliferation and and SDF-1 induced migration, that was accompanied by a concentration dependent inhibition of the MAP kinase signaling characterized by reduced phosphorylation of ERK an p38 molecules. Furthermore, the nuclear localized expression of the NF-kB members RelA and RelB was inhibited in treated cells. In addition, Syk inhibition induced apoptosis in MM cell lines and primary MM cells in a dose-dependent manner as demonstrated by flow cytometry, caspase-3 activity and PARP-1 cleavage. While there was no effect on the expression of xIAP, survivin or MCL-1, Syk inhibition reduced the expression of pro-apoptotic Bcl-2 and Bcl-xl molecules and increased the release of cytochrome c, indicating that the apoptotic cell death is mediated via the internal mitochondrial pathway. Combination of piceatannol with lenalidomide and orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235but not with bortezomib or dexamethasone enhanced the cytotoxic effects of the compound. In line with the results from previous experiments the addition of MAPK inhibitors PD98059, SP600125, U0126, SB202190 and SB203580 to piceatannol further increased the efficacy of Syk inhibition. Conclusions: Our results show that Syk inhibition might represent a promissing new treatment option in MM with an increased efficacy when combined with lenalidomide or MAP kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

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49

Flynn, Ryan, Jessica L. Allen, Leo Luznik, Kelli P. MacDonald, Katelyn Paz, Kylie A. Alexander, Ante Vulic, et al. "Targeting Syk-activated B cells in murine and human chronic graft-versus-host disease." Blood 125, no. 26 (June 25, 2015): 4085–94. http://dx.doi.org/10.1182/blood-2014-08-595470.

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50

Spurgeon, Stephen, Luke B. Fletcher, Jeffrey W. Tyner, Brian J. Druker, Anjali Pandey, Uma Sinha, and Marc M. Loriaux. "P505-15, a Highly Selective SYK Inhibitor, Shows Significant Activity In Primary CLL Cells and Is Synergistic with Fludarabine at Low Concentrations." Blood 116, no. 21 (November 19, 2010): 2839. http://dx.doi.org/10.1182/blood.v116.21.2839.2839.

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Abstract Abstract 2839 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World. Despite significant progress, CLL remains incurable so novel therapies are needed. Recent studies have highlighted the importance of B-cell receptor (BCR) associated kinases in the pathogenesis of B cell malignancies and as potential therapeutic targets. Spleen tyrosine kinase (SYK) is of particular interest as its activation results in enhanced proliferation and survival of B-cells. Analysis of non-Hodgkins lymphoma (NHL) cell lines and primary chronic lymphocytic leukemia (CLL) samples have shown that SYK is persistently phosphorylated and that SYK inhibition results in abrogation of downstream kinase activity, leading to apoptosis. The kinase inhibitor FosD (R788), which has shown clinical activity in heavily pre-treated NHL and CLL patients, exhibits inhibitory activity against SYK (IC50 = 41nM) but also inhibits a broad spectrum of other kinase targets. By contrast, P505-15 is a novel small-molecule SYK inhibitor (SYK IC50 = 1nM) that is far more selective than previously described SYK inhibitory compounds with anti SYK activity that is at least 80-fold greater than its affinity for other kinases. We applied P505-15 to primary CLL cells to evaluate the effect of P505-15 on cell viability alone or in combination with fludarabine. Methods: Fresh primary CLL cells from 42 CLL patients were purified using a Ficoll gradient. Purified cells were then added to wells (5 × 104 per well) containing four serial dilutions of P505-15 (ranging from 10 nM to 10 μM) with or without different concentrations of fludarabine (also ranging from 10nM to 10 μM). Three days after adding primary CLL cells to each well, we performed a tetrazolium-based cell viability assay (MTS) to evaluate the effect of P505-15 on CLL cells. The viability data are normalized to untreated controls and used to calculate IC50 values. Synergy (P505-15 plus fludarabine) was evaluated using the Calcusyn program. Results: We saw significantly decreased cell viability (IC50 < 3 μM) in 15/42 (36%) of primary CLL samples. Twelve (29%) of these samples had IC50 drug concentrations < 1 μM (median 393.6 nM). One of seven samples with the 17p deletion exhibited significant sensitivity (IC50 = 37.5 nM). In the presence of P505-15, significant decreases in cell viability were also seen in samples from relapsed patients and in those with additional poor risk features such as ZAP70 and/or CD38 expression, chromosome 11q deletion, and/or an unmutated IgVh. When P505-15 was combined with fludarabine (n=15), synergy was observed in 13 (87%) of samples including at the lowest concentrations of P505-15 (10 nM) and fludarabine (10 nM)-See Figure 1. Conclusions: These data demonstrate single agent activity of the highly specific SYK inhibitor P505-15 in CLL. Combination therapy with fludarabine is synergistic at very low concentrations of both P505-15 and fludarabine. Thus, P505-15 appears to be an attractive compound for clinical development especially given its high selectivity for SYK. Our results validate the rationale of targeting SYK in CLL. An initial human dose finding study in normal healthy volunteers is ongoing. Disclosures: Druker: Molecular MD: Equity Ownership. Pandey:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals: Employment.

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