Overexpression Of Syk Research Articles

Tyrobp deficiency blocks NLRP3-mediated inflammation and pyroptosis to alleviate myocardial ischemia-reperfusion injury through regulating Syk

PurposeThe present study aims to investigate the biological function of Tyrobp in myocardial ischemia-reperfusion injury (MIRI) and to clarify its potential reaction mechanisms. MethodsAC16 cells were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate the MIRI in vitro. The cell transfection technology was used to downregulate Tyrobp, followed by assessment of cell damage, apoptosis and cytokines production via Cell Counting Kit (CCK)-8 assay, lactate dehydrogenase (LDH) release assay, TUNEL and ELISA assays, respectively. Immunofluorescence assay was performed to assess GSDMD. Corresponding proteins were detected via western blotting, and Co-immunoprecipitation (Co-IP) assay was used to validate proteins interaction. ResultsTyrobp was upregulated in OGD/R-exposed AC16 cells, and Tyrobp deficiency significantly alleviated OGD/R-caused cell viability loss, LDH release and cell apoptosis in AC16 cells. Meanwhile, Tyrobp deficiency inhibited the activation of NLRP3 inflammasome, reduced the production of cytokines and inhibited GSDMD intensity and GSDMD-N expression. Additionally, Tyrobp could interact with Syk and regulate Syk/NF-κB signaling. The rescue experiments showed that the above effects of Tyrobp deficiency on OGD/R-exposed AC16 cells were partly weakened by Syk overexpression. ConclusionTyrobp deficiency alleviated MIRI by inhibiting NLRP3-mediated inflammation and pyroptosis through regulating Syk, providing a novel target for the treatment of MIRI.

Germline immunomodulatory expression quantitative trait loci (ieQTLs) associated with immune-related toxicity from checkpoint inhibition

BackgroundImmune checkpoint inhibition (ICI) has improved clinical outcomes for metastatic melanoma patients; however, 65–80% of patients treated with ICI experience immune-related adverse events (irAEs). Given the plausible link of irAEs with underlying host immunity, we explored whether germline genetic variants controlling the expression of 42 immunomodulatory genes were associated with the risk of irAEs in melanoma patients treated with the single-agent anti-CTLA-4 antibody ipilimumab (IPI). MethodsWe identified 42 immunomodulatory expression quantitative trait loci (ieQTLs) most significantly associated with the expression of 382 immune-related genes. These germline variants were genotyped in IPI-treated melanoma patients, collected as part of a multi-institutional collaboration. We tested the association of ieQTLs with irAEs in a discovery cohort of 95 patients, followed by validation in an additional 97 patients. ResultsWe found that the alternate allele of rs7036417, a variant linked to increased expression of SYK, was strongly associated with an increased risk of grade 3–4 toxicity [odds ratio (OR) = 7.46; 95% confidence interval (CI) = 2.65–21.03; p = 1.43E-04]. This variant was not associated with response (OR = 0.90; 95% CI = 0.37–2.21; p = 0.82). ConclusionWe report that rs7036417 is associated with increased risk of severe irAEs, independent of IPI efficacy. SYK plays an important role in B-cell/T-cell expansion, and increased pSYK has been reported in patients with autoimmune disease. The association between rs7036417 and IPI irAEs in our data suggests a role of SYK overexpression in irAE development. These findings support the hypothesis that inherited variation in immune-related pathways modulates ICI toxicity and suggests SYK as a possible future target for therapies to reduce irAEs.

SYK-mediated epithelial cell state is associated with response to c-Met inhibitors in c-Met-overexpressing lung cancer

Genomic MET amplification and exon 14 skipping are currently clinically recognized biomarkers for stratifying subsets of non-small cell lung cancer (NSCLC) patients according to the predicted response to c-Met inhibitors (c-Metis), yet the overall clinical benefit of this strategy is quite limited. Notably, c-Met protein overexpression, which occurs in approximately 20–25% of NSCLC patients, has not yet been clearly defined as a clinically useful biomarker. An optimized strategy for accurately classifying patients with c-Met overexpression for decision-making regarding c-Meti treatment is lacking. Herein, we found that SYK regulates the plasticity of cells in an epithelial state and is associated with their sensitivity to c-Metis both in vitro and in vivo in PDX models with c-Met overexpression regardless of MET gene status. Furthermore, TGF-β1 treatment resulted in SYK transcriptional downregulation, increased Sp1-mediated transcription of FRA1, and restored the mesenchymal state, which conferred resistance to c-Metis. Clinically, a subpopulation of NSCLC patients with c-Met overexpression coupled with SYK overexpression exhibited a high response rate of 73.3% and longer progression-free survival with c-Meti treatment than other patients. SYK negativity coupled with TGF-β1 positivity conferred de novo and acquired resistance. In summary, SYK regulates cell plasticity toward a therapy-sensitive epithelial cell state. Furthermore, our findings showed that SYK overexpression can aid in precisely stratifying NSCLC patients with c-Met overexpression regardless of MET alterations and expand the population predicted to benefit from c-Met-targeted therapy.

STAT5 Signaling Promotes Resistance to Ivosidenib in IDH1-Mutated AML

Mutations in isocitrate dehydrogenase 1 (IDH1) promote leukemic transformation through the production of an oncometabolite, 2-hydroxyglutarate (2-HG). Ivosidenib is an inhibitor of mutant IDH1 approved for the treatment of IDH1-mutated AML. Treatment with ivosidenib can induce terminal differentiation of leukemic blasts via suppression of 2-HG. However, ivosidenib as a single agent has limited efficacy, highlighting the need to better understand the mechanisms of drug resistance.The study of drug resistance mechanisms has been hindered by the lack of IDH1/2-mutated AML cell lines models. To address this issue, we derived an Idh1-mutated AML cell line from bone marrow cells of a murine AML model generated by crossing mice expressing mutant Idh1R132H with mice expressing mutant Npm1 (Npm1c). This cell line (henceforth termed “OCI-mIDH1/N”) undergoes partial myeloid differentiation in response to ivosidenib treatment in vitro.The establishment of OCI-mIDH1/N enabled us to perform a genome-wide CRISPR knockout screen to identify genes that upon inactivation, increased the differentiation response to ivosidenib. Through this screen, we identified C-type lectin member 5a (Clec5a) as one of the top hits. Clec5a encodes a cell surface receptor that signals through the intracellular spleen tyrosine kinase (SYK). To confirm this hit, we generated Clec5a knockout clones of OCI-mIDH1/N cells. Consistent with results of the screen, ivosidenib treatment induced higher levels of Gr-1, a myeloid differentiation marker, on Clec5a-/- cells than on parental Clec5a+/+ cells. Next, we investigated the role in SYK. Clec5a-/- cells had lower levels of pSYK compared with Clec5a+/+ cells, and overexpression of Syk in Clec5a-/- cells reversed their sensitization to ivosidenib. Furthermore, direct inhibition of SYK with fostamatinib was sufficient to sensitize Clec5a+/+ cells to ivosidenib. Our findings show that CLEC5A-SYK signaling promotes resistance to ivosidenib-induced differentiation.Mechanistically, we found that CLEC5A-SYK signaling drives ivosidenib resistance through STAT5 dependent expression of self-renewal genes in the HOX family. Clec5a-/- and Syk-/- OCI-mIDH1/N cells exhibited lower levels of STAT5 activation compared with wildtype cells. Furthermore, SYK inhibitor treatment downregulated pSTAT5 and decreased STAT5 occupancy at HOX gene clusters. Functionally, overexpression of constitutively active STAT5 in Clec5a-/- and Syk-/- cells reversed their heightened sensitivity to ivosidenib and increased the expression of key HOX self-renewal genes. To determine the clinical relevance of these findings, we analyzed two independent RNA-seq datasets from patients treated with IDH inhibitors (Quek et al., 2018; Wang et al., 2021). Gene set enrichment analysis revealed that poor responders expressed significantly higher levels of STAT5 target genes compared with responders in both datasets.Mutations in the receptor tyrosine kinase (RTK) pathway have previously been shown to be associated with resistance to IDH inhibitors. Given that these pathways signal through STAT5, we hypothesized that the mechanism by the RTK mutations confer ivosidenib resistance is through STAT5 activation. To test this hypothesis, we ectopically expressed KRASG12D or PTPN11E76G in OCI-mIDH1/N cells. Expression of the oncogenes increased pSTAT5 and conferred resistance to ivosidenib-induced differentiation. Importantly, knockdown of Stat5 completely restored their sensitivity to ivosidenib, in support for our hypothesis.Next, we tested if the STAT5 inhibitor pimozide synergizes with ivosidenib to treat patient-derived xenograft (PDX) models of human IDH1-mutated AML. In two PDX models where ivosidenib alone induced moderate differentiation, the addition of pimozide to ivosidenib significantly increased the expression of myeloid differentiation markers on AML cells. In two PDX models where ivosidenib alone induced no differentiation, pimozide alone was sufficient to induce profound differentiation of AML cells. Collectively, these results suggest that ivosidenib resistant cells shift their dependence from 2-HG to STAT5 to maintain their undifferentiated state.In summary, our findings demonstrate that STAT5 is a critical mediator of resistance to ivosidenib, and combination therapy with pimozide and ivosidenib is a promising therapeutic approach for IDH1-mutated AML. DisclosuresChan: BMS: Research Funding; AbbVie: Research Funding.

A TCM formula VYAC ameliorates DNCB-induced atopic dermatitis via blocking mast cell degranulation and suppressing NF-κB pathway

Ethnopharmacological relevanceA Traditional Chinese Medicine (TCM) formula (VYAC) consists of three herbs including Viola yedoensis Makino, herb (Violaceae, Viola), Sophora flavescens Aiton, root (Fabaceae, Sophora) and Dictamnus dasycarpus Turcz, root and rhizome (Rutaceae, Dictamnus), has been traditionally prescribed to treat various skin diseases in clinic. Aim of the studyThis study aims to investigate the therapeutic effects of VYAC on the 2,4-dinitrobenzene (DNCB) induced atopic dermatitis (AD)-like mice and to explore the underlying mechanisms. Materials and methodsVYAC was extracted with 70 % aqueous ethanol and lyophilized powder was used. AD-like mice were challenged by DNCB, VYAC (150 and 300 mg/kg) were oral administration daily from day 7 to day 28. At the end of experiment, the clinical scores were recorded, serum and skin in the dorsal were isolated to evaluate the therapeutic effects of VYAC. RBL-2H3 cells were stimulated with C48/80 for degranulation and plasmids expressing constitutively active form of Syk (Silence or overexpression) were transfected into RBL-2H3 cells to explore the underlying mechanisms in vitro. ResultsVYAC significantly ameliorated the cardinal symptoms in the DNCB-induced AD-like mice by repairing the skin barrier function, inhibiting mast cells infiltration, restraining the serum IgE and histamine release and decreasing TNF-α, IL-4 as well as Syk mRNA level in dorsal skin and alleviating inflammation. Besides, VYAC significantly blocked RBL-2H3 cells degranulation, reduced β-hexosaminidase and histamine release, and suppressed NF-κB pathway. What's more, the degranulation of RBL-2H3 was reduced after Syk silence and increased after Syk overexpression. ConclusionOur findings clearly suggested that VYAC treat AD through inhibiting the inflammatory mediator productions and blocking mast cell degranulation via suppressing Syk mediated NF-κB pathway.

Abstract 1257: HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors

Abstract Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduces overall survival. Approximately 30 % of AML patients carry FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutations. Sustained FLT3 inhibition can result in the emergence of resistance-conferring genetic alteration in TKD domain, usually at residues D835 and F691. Therefore, acquired TKD mutations has become a critical therapeutic target in AML therapy. On the other hand, SYK is one of kinases deeply implicated in many hematologic malignancies and highly activated in FLT3 mutation AML. SYK overexpression is known to promote over transformation of FLT3 driven AML and induce resistance to FLT3 targeted therapy. In this study, we characterized HM43239, a novel FLT3 inhibitor with SYK inhibitory activity, and assessed its potential as a novel therapeutic agent to overcome the resistances against AML therapy using current FLT3 inhibitors. HM43239 is an orally active small molecule inhibitor and it exhibited sub-nanomolar potency on binding affinity for FLT3 wild type, ITD, TKDs and ITD/TKDs mutants. HM43239 potently inhibited phosphorylation of FLT3 and its downstream such as p-STAT3/p-STAT5 dose dependently in both MOLM-14 cells harboring FLT3 ITD/F691L and FLT3 ITD/D835Y. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. Moreover, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG-1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Furthermore, HM43239 showed to be exhibited good inhibitory activity against FLT3 mutated AML cell lines and effectively regress the tumors in MOLM-14 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. On the other hand, it was confirmed that HM43239 strongly inhibited the phosphorylation of SYK in the medium of co-culture of stromal cells and AML cells. As a result, HM43239 alone more effectively induced tumor regression and prolonged the survival duration of animals than an approved FLT3 inhibitor (e.g. gilteritinib) in resistant FLT3 ITD/D835Y or ITD/F691L mutated MOLM-14 xenograft mice models. These results suggest that HM43239 could overcome the resistance induced by bone marrow microenvironment in AML patients. Taken together, HM43239 showed strong anticancer activity through various in vitro and in vivo preclinical models of AML, implicating the mechanism of overcoming resistance and preventing relapse. The effect of HM43239 in human would be demonstrated in ongoing Phase I/II clinical trials (NCT03850574) to develop promising therapeutics for patients with AML. Citation Format: Inhwan Bae, Jaeyul Choi, Jiyoung Song, Joo-Yun Byun, Eunyoung Lee, Taehun Song, Yu-Yon Kim, Yesol Bak, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1257.

Alzheimer\u2019s disease pathological lesions activate the spleen tyrosine kinase

The pathology of Alzheimer’s disease (AD) is characterized by dystrophic neurites (DNs) surrounding extracellular Aβ-plaques, microgliosis, astrogliosis, intraneuronal tau hyperphosphorylation and aggregation. We have previously shown that inhibition of the spleen tyrosine kinase (Syk) lowers Aβ production and tau hyperphosphorylation in vitro and in vivo. Here, we demonstrate that Aβ-overexpressing Tg PS1/APPsw, Tg APPsw mice, and tau overexpressing Tg Tau P301S mice exhibit a pathological activation of Syk compared to wild-type littermates. Syk activation is occurring in a subset of microglia and is age-dependently increased in Aβ-plaque-associated dystrophic neurites of Tg PS1/APPsw and Tg APPsw mice. In Tg Tau P301S mice, a pure model of tauopathy, activated Syk occurs in neurons that show an accumulation of misfolded and hyperphosphorylated tau in the cortex and hippocampus. Interestingly, the tau pathology is exacerbated in neurons that display high levels of Syk activation supporting a role of Syk in the formation of tau pathological species in vivo. Importantly, human AD brain sections show both pathological Syk activation in DNs around Aβ deposits and in neurons immunopositive for pathological tau species recapitulating the data obtained in transgenic mouse models of AD. Additionally, we show that Syk overexpression leads to increased tau accumulation and promotes tau hyperphosphorylation at multiple epitopes in human neuron-like SH-SY5Y cells, further supporting a role of Syk in the formation of tau pathogenic species. Collectively, our data show that Syk activation occurs following Aβ deposition and the formation of tau pathological species. Given that we have previously shown that Syk activation also promotes Aβ formation and tau hyperphosphorylation, our data suggest that AD pathological lesions may be self-propagating via a Syk dependent mechanism highlighting Syk as an attractive therapeutic target for the treatment of AD.

Syk Plays a Critical Role in the Expression and Activation of IRAK1 in LPS-Treated Macrophages.

To address how interleukin-1 receptor-associated kinase 1 (IRAK1) is controlled by other enzymes activated by toll-like receptor (TLR) 4, we investigated the possibility that spleen tyrosine kinase (Syk), a protein tyrosine kinase that is activated at an earlier stage during TLR4 activation, plays a central role in regulating the functional activation of IRAK1. Indeed, we found that overexpression of myeloid differentiation primary response gene 88 (MyD88), an adaptor molecule that drives TLR signaling, induced IRAK1 expression and that piceatannol, a Syk inhibitor, successfully suppressed the MyD88-dependent upregulation of IRAK1 under LPS treatment conditions. Interestingly, in Syk-knockout RAW264.7 cells, IRAK1 activity was almost completely blocked after LPS treatment, while providing a Syk-recovery gene to the knockout cells successfully restored IRAK1 expression. According to our measurements of IRAK1 mRNA levels, the transcriptional upregulation of IRAK1 was induced by LPS treatment between 4 and 60 min, and this can be suppressed in Syk knockout cells, providing an effect similar that that seen under piceatannol treatment. The overexpression of Syk reverses this effect and leads to a significantly higher IRAK1 mRNA level. Collectively, our results strongly suggest that Syk plays a critical role in regulating both the activity and transcriptional level of IRAK1.

Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling By Suppression of Mir-146a in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is driven by cell populations with stem cell-like characteristics, so called leukemia stem cells (LSC). The transcription factor Meis1 is one of the critical regulators of LSC and is capable to rapidly induce AML in murine models in the context of Hox gene overexpression. Despite sophisticated studies identifying Hox- and Meis1-regulated genes, the knowledge about their impact on intracellular signaling pathways and its functional consequences is still limited. Since Hox and Meis1 gene overexpression is often found in high risk AML and since both factors are currently considered as undruggable, we aimed to elucidate their role in regulating intracellular signaling and to investigate, if cells transformed by Hoxa9 and Meis1 are addicted to certain signaling processes.To characterize the effect of Meis1 in the context of Hox gene overexpression on protein expression and intracellular signaling, we have performed a comprehensive (phospho)proteomic analysis and correlated it with transcriptome sequencing data. Our analysis revealed that Meis1 upregulates expression of spleen tyrosine kinase (Syk) without affecting its mRNA expression level. This was confirmed in patient-derived AML cells. By global analysis of microRNA expression and subsequent functional analyses, we could identify the downregulation of miR-146a, which turned out to be PU.1-dependent, as a mediator for this post-transcriptional upregulation of SYK in Hoxa9/Meis1 overexpressing cells. To further investigate, if an activation of Syk signaling can mimic Meis1 in inducing leukemia in our murine AML transplantation model, we overexpressed Syk in Hoxa9-transformed myeloid progenitors and found that this resulted in an acceleration in leukemia development comparable to the acceleration observed upon combined Hoxa9/Meis1 overexpression. We also found that Syk overexpression resulted in an increased expression of Meis1 and an induction of Meis1-dependent gene expression signatures. Notably, Hoxa9/Meis1-transformed cells also exhibited a remarkable sensitivity to Syk inhibition and SYK knockdown in vitro and in vivo, while Hoxa9-transformed cells did not.In summary, we identified a previously unknown signaling loop between Meis1 overexpression and Syk signaling involving miR-146a as a regulator of SYK expression. Hence, we believe that Syk inhibition by small molecules might be a potential therapeutic option for AMLs particularly in the context of Hox/Meis1-overexpression. DisclosuresBerg:Astellas: Other: Travel Funding; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding.

Harnessing Negative B Cell Selection to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Background: B cells are selected for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: attenuation below minimum (e.g. non-functional BCR) or hyperactivation above maximum (e.g. autoreactive BCR) thresholds of signaling strength causes negative selection and cell death. About 25% of B cell acute lymphoblastic leukemia (ALL) is driven by oncogenic tyrosine kinases (e.g. BCR-ABL1 in Ph+ ALL), which mimics constitutively active pre-BCR signaling and defines the ALL subgroup with the worst clinical outcome. Currently more potent tyrosine kinase inhibitors (TKI) are developed for Ph+ ALL to suppress oncogenic signaling below a minimum threshold for survival. However Ph+ ALL cells invariably develop resistance against TKI. Here, we tested the hypothesis that targeted hyperactivation of oncogenic signaling above a maximum threshold will trigger B cell-inherent mechanisms of negative selection and selectively kill Ph+ALL cells.Results: The Ph+ ALL cells don not express a functional pre-BCR and BCR-ABL1 oncogene mimics a constitutively active pre-BCR by phosphorylating SYK, LYN, BTK and PLCg2. An incremental increase of pre-BCR downstream signaling (ITAM or SYK overexpression) was indeed sufficient to induce cell death in Ph+ ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ALL cells in this experimental setting.Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1 (CD31), CD300A and LAIR1. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In the ECOG trial (E2993; n=215) for adults ALL, PECAM1 mRNA level negatively correlated with OS rate (p=0.0285).Genetic studies revealed that Pecam1, Cd300a and Lair1 receptors are critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (SHP1) and Inpp5d (SHIP1). Deletion of Pecam1, Cd300a or Lair1 in Ph+ ALL cells caused increased ROS levels, G0/G1cell cycle arrest, decreased colony formation capacity and cellular senescence. Phosphorylation of pre-BCR downstream molecules (SYK, LYN, BTK and PLCg2) was increased after Lair1 deletion and this hyper-signaling could not be tolerated by Ph+ ALL cells. Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice in a transplant experiment. Leukemia cell death caused by Lair1-deletion could be rescued by overexpression of the active inhibitory phosphatase Ptpn6 (CD8-SHP1) or Inpp5d (CD8-SHIP1). Genetic deletion of Ptpn6 and Inpp5d caused increased pre-BCR signaling and cell death in BCR-ABL1 ALL cells but not myeloid cells (normal and BCR-ABL1-transformed), which -unlike B cells- are not subject to negative selection of auto-reactive clones. Decreasing pre-BCR signaling by SYK inhibition rescued cell death after Ptpn6- or Inpp5d- deletion.Blocking inhibitory receptors by using chimeric PECAM1, CD300A and LAIR1 receptor decoys inhibited proliferation and caused cell death in Ph+ ALL xenograft cells. More potently, a novel small molecule inhibitor of INPP5D (SHIP1) selectively killed Ph+ ALL xenograft cells through inducing hyper pre-BCR signaling, regardless of TKI resistance. We demonstrate that inhibitory phosphatase signaling represents a potential novel class of therapeutic targets for Ph+ALL.Conclusions: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified ITIM-receptors and phosphatases as members of a potential novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

In vivo and in vitro anti-inflammatory activities of Persicaria chinensis methanolic extract targeting Src/Syk/NF-κB

Ethnopharmacologic relevancePersicaria chinensis L. (Polygonaceae) [also synonym as Polygonum chimnense L.] has been used as Chinese traditional medicine to treat ulcer, eczema, stomach ache, and various inflammatory skin diseases. Due to no molecular pharmacological evidence of this anti-inflammatory herbal plant, we investigated the inhibitory mechanisms and target proteins contributing to the anti-inflammatory responses of the plant by using its methanolic extract (Pc-ME). Materials and methodsWe used lipopolysaccharide (LPS)-treated macrophages and a murine HCl/EtOH-induced gastritis model to evaluate the anti-inflammatory activity of Pc-ME. HPLC analysis was employed to identify potential active components of this extract. Molecular approaches including kinase assays, reporter gene assays, immunoprecipitation analysis, and overexpression of target enzymes were used to confirm target enzymes. ResultsPc-ME inhibited LPS-induced nitric oxide and prostaglandin E2 release by RAW264.7 macrophages and ameliorated HCl/EtOH-induced gastric ulcers in mice. The nuclear translocation of NF-κB (p65 and p50) was suppressed by Pc-ME. Phosphorylation of Src and Syk, their kinase activities, and formation of the signaling complex of these proteins were repressed by Pc-ME. Phosphorylation of p85 and Akt induced by Src or Syk overexpression was blocked by Pc-ME. In the mouse gastritis model, orally administered Pc-ME suppressed the increased phosphorylation of IκBα, Αkt, Src, and Syk. Caffeic acid, kaempferol, and quercetin, identified as major anti-inflammatory components of Pc-ME by HPLC, displayed strong nitric oxide inhibitory activity in LPS-treated macrophages. ConclusionPc-ME might play a pivotal ethnopharmacologic role as an anti-inflammatory herbal medicine by targeting Syk and Src kinases and their downstream transcription factor NF-κB.

(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl 6-hydroxynaphthalene-2-carboxylate, a kojic acid derivative, inhibits inflammatory mediator production via the suppression of Syk/Src and NF-κB activation

Numerous derivatives of kojic acid have been synthesised to expand its immunopharmacological uses. Kojic acid is known to have anti-cancer, anti-inflammatory, and anti-melanogenesis effects. We found that (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl 6-hydroxynaphthalene-2-carboxylate (MHNC) strongly suppressed the production of nitric oxide (NO) in an initial screening experiment. In this study, we explored the in vitro and in vivo anti-inflammatory activity of MHNC and its inhibitory mechanisms using lipopolysaccharide (LPS)-treated RAW264.7 cells and HCl/EtOH-treated ICR mice. MHNC dose-dependently diminished the secretion of nitric oxide (NO) and prostaglandin (PG)E2 in LPS-treated RAW264.7 cells. This compound also suppressed the upregulation of mRNA levels for the inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 genes. Additionally, the transcriptional activation of these genes was inhibited by MHNC through the suppression of the nuclear translocation of nuclear factor (NF)-κB subunits (p65 and p50), as determined by a luciferase reporter assay. Interestingly, MHNC treatment was found to suppress a series of upstream signalling cascades consisting of IκBα, AKT, PDK1, Src, and Syk for NF-κB activation. Furthermore, a direct enzyme assay with purified Src and Syk and luciferase assays using Src and Syk overexpression indicated that these enzymes were directly inhibited by MHNC. Finally, MHNC (20mg/kg) prevented inflammatory symptoms of the stomach in mice treated with HCl/EtOH by reducing phospho-IκBα levels. Taken together, our data suggest that MHNC may negatively modulate in vitro and in vivo inflammatory responses via the direct suppression of Syk/Src and NF-κB.

Spleen Tyrosine Kinase (Syk) Regulates Systemic Lupus Erythematosus (SLE) T Cell Signaling

Engagement of the CD3/T cell receptor complex in systemic lupus erythematosus (SLE) T cells involves Syk rather than the zeta-associated protein. Because Syk is being considered as a therapeutic target we asked whether Syk is central to the multiple aberrantly modulated molecules in SLE T cells. Using a gene expression array, we demonstrate that forced expression of Syk in normal T cells reproduces most of the aberrantly expressed molecules whereas silencing of Syk in SLE T cells normalizes the expression of most abnormally expressed molecules. Protein along with gene expression modulation for select molecules was confirmed. Specifically, levels of cytokine IL-21, cell surface receptor CD44, and intracellular molecules PP2A and OAS2 increased following Syk overexpression in normal T cells and decreased after Syk silencing in SLE T cells. Our results demonstrate that levels of Syk affect the expression of a number of enzymes, cytokines and receptors that play a key role in the development of disease pathogenesis in SLE and provide support for therapeutic targeting in SLE patients.

Attenuated Natural Killer (NK) Cell Activation through C-type Lectin-like Receptor NKp80 Is Due to an Anomalous Hemi-immunoreceptor Tyrosine-based Activation Motif (HemITAM) with Impaired Syk Kinase Recruitment Capacity

Cellular cytotoxicity is the hallmark of NK cells mediating both elimination of virus-infected or malignant cells, and modulation of immune responses. NK cytotoxicity is triggered upon ligation of various activating NK cell receptors. Among these is the C-type lectin-like receptor NKp80 which is encoded in the human Natural Killer Gene Complex (NKC) adjacent to its ligand, activation-induced C-type lectin (AICL). NKp80-AICL interaction promotes cytolysis of malignant myeloid cells, but also stimulates the mutual crosstalk between NK cells and monocytes. While many activating NK cell receptors pair with ITAM-bearing adaptors, we recently reported that NKp80 signals via a hemITAM-like sequence in its cytoplasmic domain. Here we molecularly dissect the NKp80 hemITAM and demonstrate that two non-consensus amino acids, in particular arginine 6, critically impair both hemITAM phosphorylation and Syk recruitment. Impaired Syk recruitment results in a substantial attenuation of cytotoxic responses upon NKp80 ligation. Reconstituting the hemITAM consensus or Syk overexpression resulted in robust NKp80-mediated responsiveness. Collectively, our data provide a molecular rationale for the restrained activation potential of NKp80 and illustrate how subtle alterations in signaling motifs determine subsequent cellular responses. They also suggest that non-consensus alterations in the NKp80 hemITAM, as commonly present among mammalian NKp80 sequences, may have evolved to dampen NKp80-mediated cytotoxic responses toward AICL-expressing cells.

Hypermethylation and prognostic implication of Syk gene in human colorectal cancer

The study was aimed to investigate the relationship between hypermethylation of Syk gene and clinicopathological characteristics and long-term outcomes in colorectal cancer. The effect of Syk on cell proliferation and invasion ability was also assessed. Methylation and expression status of Syk were explored in CRC tissues and cell lines by MSP, qRT-PCR and western blot assay. The effects of Syk overexpression on tumorigenesis were studied by in vitro assay. The correlation between Syk methylation and clinical relevance in CRC patients was also analyzed. Syk methylation was found 48.6 % in CRC tissue samples and 57.1 % in cell lines, respectively. The loss of Syk expression could be restored by demethylation agent. Overexpression of Syk in CRC cell inhibited cell proliferation (p < 0.01) and invasion (p < 0.01). The methylation of Syk was significantly associated with histological grade (p = 0.002), lymph node status (p < 0.001) and TNM stage (p < 0.001). Five-year overall survival in methylated Syk group was significantly lower than that in unmethylated Syk group (59 vs. 80 %, p < 0.001). Multivariate analysis demonstrated that Syk methylation was an independent prognostic factor for overall survival. Syk is identified as a potential tumor suppressor in CRC progression. Syk methylation is correlated with poor overall survival, which acts as an independent prognostic indicator of CRC.

SYK expression in metastatic retinoblastoma.

e13544 Background: Retinoblastoma is an aggressive childhood cancer of the retina. While more than 90% of patients are cured when tumor is confined to the globe, for up to half of children in developing countries with advanced tumor at diagnosis, retinoblastoma remains a lethal disease. Recent whole-genome sequencing demonstrated that the retinoblastoma genome is relatively stable (Zhang et al). However, RB1 loss is associated with epigenetic deregulation of several cancer pathways. Significantly, the proto-oncogene SYK, spleen tyrosine kinase, which has no role in normal eye development, is upregulated in retinoblastoma and required for tumor cell survival (Zhang et al). Immunohistochemical (IHC) studies showed that SYK is ubiquitously overexpressed in intraocular retinoblastoma, whereas it is absent in normal retina. The purpose of this study is to investigate whether SYK overexpression persists in metastatic retinoblastoma. Methods: From 1962-2011, seven metastatic retinoblastoma tumor specimens (excluding bone marrow or cerebrospinal fluid) from six retinoblastoma patients were retrieved from the Pathology Archives in our institution. Morphology and supportive IHC, to include immunopositivity for synaptophysin and immunonegativity for cytokeratins, confirmed the diagnosis of metastatic retinoblastoma in each case. IHC for SYK was performed according to the previously published technique (Zhang et al). Results: Tumors occurred in two male and four female patients with seven metastatic retinoblastoma samples in scalp, lymph node, neck, ovary and bone (femur). By IHC, all seven metastatic retinoblastoma tumor samples were positive for SYK. Conclusions: This is the first report of SYK overexpression in metastatic retinoblastoma. Drugs targeting SYK are already in clinical trials for adults with leukemia and rheumatoid arthritis, and preclinical efficacy in vitro and in vivo has been established. Moving from bench to bedside with this epigenetically targeted agent warrants further investigation.

Abstract 1240: The role of hematopoietic protein kinases in GBM

Abstract Glioblastoma multiforme (GBM) is the most common and deadly form of brain cancer. Despite intensive research the median survival of GBM remains at 1 year, with less than 10 % of patients surviving over 5 years. Our analysis of fresh GBM, secondary GBM, high grade astrocytoma, oligodendroglioma, normal brain and normal human astrocytes showed increased expression of 19 novel protein kinases in GBM and targets previously associated with gliomagenesis (e.g. EGFR or PDGF). Two of these kinases which are mainly expressed in hematopoietic cells were the cytoplasmic kinases SYK and LYN. We confirmed the overexpression in GBM tumours and cell lines through western blot and immunohistochemistry analysis. More importantly, SYK expression was derived from the GBM tumours and not from infiltrating leukocytes as shown through colocalization with GFAP, and not with CD45. SYK was found strongly activated at the membrane after EGF and treatment with three specific small molecule inhibitors and siRNA targeting SYK strongly blocked proliferation and migration of GBM cells. This was confirmed through wtSYK and kinase dead SYK overexpression. In addition, SYK phosphorylation was found to be cell cycle regulated after synchronization. Due to our preliminary results we will test the therapeutic potential of SYK inhibitors and epistatic kinase interactions on our xenograft model measuring the efficacy of therapeutics crossing the blood brain barrier with the ultimate goal of discovering alternative treatments for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1240. doi:1538-7445.AM2012-1240

Involvement of the Syk-mTOR Pathway in Follicular Lymphoma Cell Invasion and Angiogenesis,

Abstract 3656Follicular lymphoma is the second most common non-Hodgkin's lymphoma. The disease affects the lymph nodes and 50% of patients present with bone marrow infiltration, however the mechanisms involved in dissemination of the disease are not yet known. We previously reported that follicular lymphoma cells display an over-expression of Syk, a tyrosine kinase involved in many cellular processes including cell migration. Therefore we sought to explore its role in the invasive process. Here we show that follicular lymphoma patients display higher MMP-9 and VEGF levels than healthy donors. Moreover, using Syk siRNA and the Syk inhibitor R406, we demonstrate that, in follicular lymphoma cells, Syk is involved in the regulation of MMP-9 and VEGF expression, and that invasion and angiogenesis is mediated through a PI3K-mTOR module. Finally, using a follicular lymphoma xenograft mouse model we observe that R406, the active metabolite of fostamatinib (R788), inhibits MMP-9 expression and angiogenesis in vivo. Altogether, this study provides strong evidence that Syk represents an encouraging therapeutic target in follicular lymphoma and suggests the potential use of fostamatinib as an anti-invasive and anti-angiogenic drug. Disclosures:No relevant conflicts of interest to declare.

Involvement of the Syk–mTOR pathway in follicular lymphoma cell invasion and angiogenesis

Follicular lymphoma (FL) is the second-most common non-Hodgkin's lymphoma. The disease affects the lymph nodes, and 50% of patients present with bone marrow infiltration; however, the mechanisms involved in dissemination of the disease are not yet known. We previously reported that FL cells display an overexpression of Syk, a tyrosine kinase involved in many cellular processes including cell migration. Therefore, we sought to explore its role in the invasive process. Here, we show that FL patients display higher matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF) levels than healthy donors. Moreover, using Syk small interfering RNA and the Syk inhibitor R406, we demonstrate that, in FL cells, Syk is involved in the regulation of MMP-9 and VEGF expression, and that invasion and angiogenesis is mediated through a phosphatidylinositol-3 kinase (PI3K)-mammalian target of rapamycin module. Finally, using a FL xenograft mouse model we observe that fostamatinib (R788), inhibits MMP-9 expression and angiogenesis in vivo. Altogether, this study provides strong evidence that Syk represents an encouraging therapeutic target in FL and suggests the potential use of fostamatinib as an anti-invasive and anti-angiogenic drug.

Heterogeneous expression of Src tyrosine kinases Lyn, Fyn and Syk in classical Hodgkin lymphoma: prognostic implications

The aim of this study was to determine the prognostic significance of the expression of Lyn, Fyn and Syk in Hodgkin lymphoma and its correlation with Epstein–Barr virus (EBV) infection. With this in mind, 96 patients with classical Hodgkin lymphoma were immunohistochemically evaluated for Lyn, Fyn and Syk expression in Hodgkin and Reed–Sternberg cells, and the results were correlated with the presence of EBV and patient outcomes. These three kinases were heterogeneously expressed in classical Hodgkin lymphoma cases. As there are no cut-offs established for these antibodies, they were introduced as continuous variables in the model. Statistical analysis showed that the expression of Syk and Fyn was significantly associated with shorter failure-free survival. Syk and Fyn may be useful to predict at diagnosis the treatment response of patients with classical Hodgkin lymphoma. There was a significant association between EBV infection and Lyn expression (p < 0.05). Overexpression of Syk and the availability of Syk inhibitors suggest that this molecule might be a therapeutic strategy worthy of development for cases expressing this molecule.