Spleen Tyrosine Kinase Protein Research Articles

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2+ influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca ++ influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2+ flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70 . Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

Bioactives and metabolites of Tetrastigma hemsleyanum root extract alleviate DSS-induced ulcerative colitis by targeting the SYK protein in the B cell receptor signaling pathway

Tetrastigma hemsleyanum is an endemic Chinese herb with a wide range of pharmacological activities, including anti-inflammatory, antiviral, antioxidant, antitumor, and immunomodulatory activities. However, the effect and mechanisms of the anti-inflammatory activity of T. hemsleyanum root extract against dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) have not yet been fully investigated. This study aimed to explore the therapeutic effect and molecular mechanisms of T. hemsleyanum root extract in DSS-induced UC mice and knockdown cells. T. hemsleyanum root extract was obtained and analyzed by high-performance liquid chromatography (HPLC). The therapeutic effects of T. hemsleyanum root extract on DSS-induced UC mice were evaluated by the disease activity index (DAI) score, colon length, serum inflammatory cytokines and oxidant/antioxidant levels, and histopathological features of the ileum and colon. Genome-wide gene expression profiles of ileal and colonic tissues were collected by transcriptomics, and signaling pathways were analyzed by the KEGG database. UC-related pathways were uploaded to the STRING database, then the protein-protein interactions (PPIs) were determined by Cytoscape, and the enriched genes were evaluated by real-time quantitative PCR (qPCR). The protein-ligand complexes were docked by AutoDock, and the genes were knocked down in Caco-2cells by shRNA. The non-targeted metabolomic profiling of ileal contents was analyzed by ultra-high-performance liquid chromatography (UHPLC), and gut microflora were sequenced by an Illumina MiSeq System. Ten components that alleviated UC symptoms in mice by decreasing the DAI and serum inflammatory cytokines and oxidant levels, promoting intestinal development, and increasing serum antioxidant levels were identified in T. hemsleyanum root extract. T. hemsleyanum root extract activated the B cell receptor signaling pathway in the colon tissue of UC mice, in which two components, rutin and astragaline, bound to the spleen tyrosine kinase (SYK) protein but also restored gut microflora diversity and increased the proportion of probiotics. Furthermore, metabolites of T. hemsleyanum root extract were involved in vitamin metabolism, fatty acid metabolism, and ferroptosis. The rutin and astragaline components of T. hemsleyanum root extract, by binding to SYK protein, activated the B cell receptor signaling pathway and restored gut microflora diversity to alleviate UC symptoms in mice.

Enhanced Efficacy of the SYK Inhibitor Entospletinib and Chemotherapy in Infant Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A -rearranged (R) B-cell acute lymphoblastic leukemia (B-ALL), who have extremely poor event-free survival (EFS). Addition of kinase inhibitors to chemotherapy has markedly improved outcomes for patients with some genetic subsets of B-ALL, such as BCR-ABL1 -R (Ph+) ALL. Unfortunately, addition of the FMS-like tyrosine kinase 3 (FLT3) inhibitor lestaurtinib did not improve 3-year EFS for infants with KMT2A -R and FLT3-overexpressing B-ALL in the Children's Oncology Group trial AALL0631 (Brown et al, SIOP 2016), indicating continued need for alternative therapeutic strategies. We recently observed constitutive phosphorylation (p) of spleen tyrosine kinase (SYK) at tyrosine 323 in infant ALL specimens (Figure 1A) and hypothesized that activated signaling is targetable with the selective SYK inhibitor entospletinib (ENTO). We further hypothesized that combining ENTO with chemotherapy would have superior activity in comparison to single-agent treatments.Methods: To test these hypotheses, we established patient-derived xenograft (PDX) models of KMT2A -R (n=7) and non- KMT2A -R (n=4) infant ALL in NSG mice using diagnostic and relapse bone marrow specimens from patients treated on or as per the AALL0631 trial protocol. Total and phosphorylated (p) SYK and other phosphoprotein levels were assessed by Simple WesternTM analysis of splenic lysates from ALL-engrafted PDX mice to identify leukemias with constitutive SYK signaling activation. Infant ALL PDX models were then treated with control mouse chow or chow containing 0.07% ENTO ad libitum, 0.25 mg/kg vincristine (VCR) dosed intraperitoneally once weekly, or a combination of ENTO and VCR for up to 28 days to determine treatment efficacy. Cohorts of additional infant ALL PDX mice were treated for 72 hours to assess plasma levels and pharmacodynamic effects of ENTO administration.Results: Constitutive SYK signaling was observed in several models of de novo KMT2A -R and non- KMT2A -R B-ALL. ENTO treatment of KMT2A -R ALL PDX models ALL3103 with t(9;11) and ALL135 with t(11;19) inhibited leukemic cell burden in vivo versus control animals by flow cytometric and immunohistochemical (IHC) analyses of murine spleens (representative data in Figure 1B). Importantly, combined ENTO and VCR treatment resulted in a greater reduction in ALL cell number, which was confirmed by human CD19+ IHC analysis of murine spleens and bone marrow. ENTO concentrations were maintained throughout study duration with terminal free fraction plasma levels of 61 and 230 nM (ALL3103) and 89 and 214 nM (ALL135) for ENTO and ENTO/VCR treatment arms, respectively. Evaluation of SYK pathway modulation in vivo demonstrated inhibition of pSYKY323 in human ALL cells within murine spleens that correlated with the level of ENTO exposure of treated mice (ALL3103 PDX; Spearman correlation of -0.63, p<0.05). No alterations in total SYK protein were observed. RNA sequencing of ALL3103 PDX tissues showed that ENTO treatment was associated with downregulation of MYC and of cell cycle and DNA damage gene expression signatures.Conclusions: Constitutive activation of SYK signaling occurs in infant KMT2A -R B-ALL. Entospletinib treatment of infant B-ALL PDX models potently inhibited the SYK pathway and decreased leukemic cell burden in vivo . Superior anti-ALL activity was observed with combined ENTO and VCR compared to ENTO alone. Additional studies are ongoing to determine the activity of ENTO in non- KMT2A -R and other ALL PDX models. [Display omitted] DisclosuresYahiaoui:Gilead Sciences, Inc.: Employment, Equity Ownership. Lopez:Gilead Sciences, Inc.: Employment, Equity Ownership. Mikaelian:Gilead Sciences, Inc.: Employment, Equity Ownership. Tannheimer:Gilead Sciences, Inc.: Employment, Equity Ownership. Forslund:Gilead Sciences, Inc.: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Pathological predictive factors for late recurrence of hepatocellular carcinoma in chronic liver disease.

Late recurrence of hepatocellular carcinoma (HCC) is regarded as de novo HCC from chronic hepatitis. This study investigated clinicopathological and molecular factors to develop a nomogram for predicting late HCC recurrence (>2years after curative resection). The training and validation cohorts included HCC patients with a major aetiology of hepatitis B who underwent curative resection. Clinicopathological features including lobular and porto-periportal inflammatory activity, fibrosis and liver cell change were evaluated. Proteins encoded by genes related to late recurrence were identified using a reverse phase protein array of 95 non-tumourous liver tissues. Immunoexpression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), plasminogen activator inhibitor-1, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and spleen tyrosine kinase (SYK) was measured. Late recurrence occurred in 74/402 (18%) and 47/243 (19%) in the training and validation cohorts respectively. Cirrhosis, moderate/severe lobular inflammatory activity, and expression of pSTAT3, pERK1/2, and SYK proteins correlated to the gene signature of hepatocyte injury and regeneration were independently associated with late recurrence, with odds ratios (95% confidence intervals) of 2.0 (1.2-3.3), 21.1 (4.3-102.7) and 6.0 (2.1-17.7) respectively (P<.05 for all). A nomogram based on these variables (histological parameters and immunohistochemical marker combinations) showed high reliability in both the training and validation cohorts (Harrell's C index: 0.701 and 0.716; 95% confidence intervals: 0.64-0.76 and 0.64-0.79 respectively). The combination of pSTAT3, pERK1/2 and SYK immunoexpression with high lobular inflammatory activity and cirrhosis (fibrosis) predicts late HCC recurrence.

Spleen tyrosine kinase expression is correlated with human papillomavirus in head and neck cancer.

Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC. SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models. Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p<0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models. HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.

Identifying regulatory pathways of spleen tyrosine kinase expression in human basophils

Expression levels of spleen tyrosine kinase (SYK), a critical signaling tyrosine kinase in basophils, are uniquely low relative to all other circulating leukocytes, and levels are highly variable in the population. We sought to determine whether transcriptional regulation of SYK through unique silencing of the SYK gene determines its basophil-specific expression patterns. Culture-derived basophils (CD34B cells) were derived from cultures of CD34+ progenitor cells by using 2 methods (G1 or G3). Peripheral blood basophils (PBBs; relative SYK protein level= 1), B cells (SYK= 8), CD34B-G1 cells (SYK= 11), and CD34B-G3 cells (SYK= 5) were examined by using assay for transposase-accessible chromatin sequencing (ATAC-seq) methods. In addition, the transcriptomes of 6 cell types, PBBs, peripheral blood eosinophils (SYK= 11), plasmacytoid dendritic cells (SYK= 30), CD34+ progenitors (SYK= 11), CD34B-G1 cells, and CD34B-G3 cells, were analyzed for patterns that matched patterns of SYK expression in these cells, with a focus on transcription factors. ATAC-seq showed that PBBs have multiple open regions in the SYK gene, suggesting a nonsilenced state with 1 region unique to PBBs (low SYK expression), 1 region unique to both PBBs (low SYK expression) and both G1 and G3 CD34B cells (high and moderate SYK expression, respectively), and 5 regions unique to B cells (high SYK expression). SYK expression across the 6 cell types explored showed a unique pattern that was matched to expression patterns of 3 transcription factors: Kruppel-like factor 5 (KLF5), zinc-finger protein 608 (ZNF608), and musculoaponeurotic fibrosarcoma protein (c-MAF). Two new potential regulatory pathways for SYK expression were identified. One appears independent of transcriptional regulation, and one appears to be dependent on transcriptional control in the SYK gene.

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

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

SYK Inhibition Potentiates the Effect of Chemotherapeutic Drugs on Neuroblastoma Cells in Vitro.

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 61-3606 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.

Cooperation between SYK and ZAP70 Kinases As a Driver of Oncogenic BCR-Signaling in B-Cell Malignancies

The spleen tyrosine kinase (SYK) and ζ-associated protein of 70 kD (ZAP70) tyrosine kinases play critical roles in proximal signal transduction of B-cell (BCR) and T-cell receptors (TCR), respectively. The highly similar SYK and ZAP70 kinases share a common structure composed of two tandem SH2 domains and a carboxy-terminal kinase domain. A linker region, termed interdomain B, connects the SH2 domains to the kinase domain and is important for kinase activation. Despite their conserved structure, SYK and ZAP70 are expressed in a largely mutually exclusive manner and play analogous roles in BCR- and TCR-signaling.Cross-lineage activation of ZAP70 in B cells was previously identified in chronic lymphocytic leukemia (CLL), which is characterized by clonal accumulation of malignant CD5+ B-cell cells that retain dependency on the BCR for survival signals. Nearly half of CLL cases show co-expression of SYK and ZAP70, and these patients have an aggressive disease course and a poor prognosis.Our analysis shows that in addition to CLL, aberrant ZAP70 expression occurs in other B-cell malignancies, e.g. TCF3-PBX1 pre-B ALL and B-cell lymphoma subsets that depend on survival signals from a functional (pre-) BCR. These findings suggest that interactions between SYK and ZAP70 may function to fine-tune strength of oncogenic BCR-signaling.To test this hypothesis, we have used a combination of molecular and proteomic approaches. We studied mechanisms by which ZAP70 integrates into BCR-mediated signals, and how the function of ZAP70 in B-cells differs from its native role downstream of the TCR. We demonstrate that ectopically expressed SYK and ZAP70 proteins are constitutively phosphorylated in BCR-ABL1+ B-ALL cells, but these induce distinctive signaling thresholds. CRISPR-mediated deletion of SYK or ZAP70 in leukemic cells further revealed that SYK and ZAP70 regulate unique signaling pathways in B-cells.We also demonstrate that ZAP70 is activated following BCR stimulation of lymphoma cells, and SYK/ZAP70 co-expressing cells display enhanced BCR signaling. Interestingly, enhanced BCR signaling was also observed in cells engineered to express an alternative splice variant of SYK (SYK-S). This shorter isoform of SYK, lacks a 23 amino-acid insert in the interdomain-B linker region, which is also absent in ZAP70, and may define unique protein-interactions that modulate signaling outcome. To elucidate the differential interactome of SYK, SYK-S, and ZAP70 we performed proximity-dependent biotin identification (BioID) experiments in B-cells following BCR-activation to capture the core signalling networks of these kinases in leukemic cells. In addition to expected BCR components including BLNK, PTPN6 and CBL we identified novel SYK and ZAP70 associated molecules including IKZF3, LAT2 and WAS which may play important roles in the survival of BCR-dependent malignancies. Importantly our findings highlight a role for ZAP70 in oncogenic BCR-signaling and suggest that ZAP70 promotes oncogenic BCR-signaling by limiting the ability of the BCR to induce negative B-cell selection and cell death. DisclosuresNo relevant conflicts of interest to declare.

Tyrosine kinase SYK is a potential therapeutic target for liver fibrosis.

Spleen tyrosine kinase (SYK) plays a critical role in immune cell signaling pathways and has been reported as a biomarker for human hepatocellular carcinoma (HCC). We sought to investigate the mechanism by which SYK promotes liver fibrosis and to evaluate SYK as a therapeutic target for liver fibrosis. We evaluated the cellular localization of SYK and the association between SYK expression and liver fibrogenesis in normal, hepatitis B virus (HBV)-infected, hepatitis C virus (HCV)-infected and non-alcoholic steatohepatitis (NASH) liver tissue (n=36, 127, 22 and 30, respectively). A polymerase chain reaction (PCR) array was used to detect the changes in transcription factor (TF) expression in hepatic stellate cells (HSCs) with SYK knockdown. The effects of SYK antagonism on liver fibrogenesis were studied in LX-2 cells, TWNT-4 cells, primary human HSCs, and three progressive fibrosis/cirrhosis animal models, including a CCL4 mouse model, and diethylnitrosamine (DEN) and bile duct ligation (BDL) rat models. We found that SYK protein in HSCs and hepatocytes correlated positively with liver fibrosis stage in human liver tissue. HBV or HCV infection significantly increased SYK and cytokine expression in hepatocytes. Increasing cytokine production further induced SYK expression and fibrosis-related gene transcription in HSCs. Up-regulated SYK in HSCs promoted HSC activation by increasing the expression of specific TFs related to activation of HSCs. SYK antagonism effectively suppressed liver fibrosis via inhibition of HSC activation, and decreased obstructive jaundice and reduced HCC development in animal models. Conclusion: SYK promotes liver fibrosis via activation of HSCs and is an attractive potential therapeutic target for liver fibrosis and prevention of HCC development. (Hepatology 2018).

Activated spleen tyrosine kinase promotes malignant progression of oral squamous cell carcinoma via mTOR/S6 signaling pathway in an ERK1/2-independent manner.

Spleen tyrosine kinase (SYK), a non-receptor cytoplasmic tyrosine enzyme, is well known for its ability in certain pathways through immune receptors. Recently, SYK role in cancer has been widely studied. SYK plays a dual role as a tumor suppressor and tumor promoter. Nevertheless, its role in oral squamous cell carcinoma (OSCC) has not been fully investigated. In the current study, samples from OSCC tumors and adjacent normal counterparts were collected and SYK expression was evaluated by real-time qPCR. SYK mRNA expression in tumors was higher than the normal tissues. And high SYK expression was confirmed by immunohistochemistry analysis and closely related to worse overall survival. The expression of SYK mRNA and protein was detected in 2 of 4 OSCC cell lines. SYK pharmacological suppression and RNAi-mediated knockdown inhibited proliferation, migration, and invasion of SYK-positive cells by reducing phosphorylated ERK1/2 and mTOR levels. One inhibitor of MEK, PD98059, also suppressed the same cancer-associated phenotypes of SYK-positive cells by decreasing phosphorylated ERK1/2 but increasing phosphorylated mTOR. Piceatannol, one pharmacological inhibitor of SYK, attenuated tumor growth in vivo. Overall, our results revealed a novel mechanism triggered by SYK to increase OSCC tumoriogenesis and tumor progression.

Abstract 2465: Spleen tyrosine kinase (SYK) in neuroblastoma tumorigenesis

Abstract Tyrosine kinases play an important role in tumorigenesis by contributing to the regulation of cell proliferation and survival, and are therefore among the most attractive drug targets. SYK is a non-receptor tyrosine kinase highly expressed in cells of hematopoietic origin where it contributes to immune receptor signaling. The role of SYK in cancer is best understood in hematological malignancies where it promotes survival, and SYK inhibition has been shown to be a promising therapeutic approach. For solid tumors the role of SYK remains double-edged, as it has been found to promote but also suppress tumor development dependent on the cancer type. Besides its role in survival signaling SYK has also been found to contribute to drug resistance in several malignancies. Neuroblastoma (NB) is a malignancy of early childhood. Although there have been great advances in the treatment of NB during the past 30 years, the prognosis for high- risk patients remains poor. In this study we have evaluated SYK as a potential drug target in NB. Examination of gene expression datasets indicated a correlation between high expression of SYK and poor prognosis in NB. Screening of NB cell lines showed that SYK mRNA was present in the majority of NB cells lines whereas the SYK protein was detected less frequently. However, SYK was detectable in NB tumor tissue using immunohistochemistry. The efficacy of different, commercially available, SYK inhibitors was evaluated by comparing the neuroblastoma cell lines SH-SY5Y (high SYK expression) and SK-N-BE(2) (low SYK expression) using a cell viability assay. While one inhibitor displayed a markedly stronger effect on the viability of SH-SY5Y cells compared to SK-N-BE(2) the effect of the other inhibitors appears to be less specific. To confirm that SYK inhibition leads to decreased cell proliferation SYK specific siRNA was used. After 48h incubation the cell viability of SH-SY5Y cells was decreased by approximately 25%. Our findings indicate that SYK may be a possible drug target in neuroblastoma. To further evaluate its potential, combination studies using SYK inhibitors and cytostatic drugs will be performed. Citation Format: Conny Tuemmler, Gianina Dumitriu, Julia Cserna, Ugo L. Moens, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. Spleen tyrosine kinase (SYK) in neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2465.

C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits

C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities invitro and expression of epithelial to mesenchymal transition markers; these tumor-suppressive effects of CLEC2 required SYK. CLEC2 and SYK interacted physically, and SYK maintained the stability of CLEC2 in cells. AGS cells with CLEC2 knockdown had increased levels of phosphorylated AKT and glycogen synthase kinase-3 beta, increased expression of Snail, reduced levels of E-cadherin, and formed more metastases in mice than AGS cells that expressed CLEC2; these knockdown changes were prevented by the PI3K inhibitor LY294002. Activation of CLEC2 in AGS cells reduced protein and messenger RNA levels of PI3K subunits p85 and p110; this effect was blocked by SYK inhibitor R406. Levels of CLEC2 and SYK proteins and messenger RNAs correlated in gastric tumor samples. CLEC2 suppresses metastasis of gastric cancer cells injected into mice, and prevents activation of AKT and glycogen synthase kinase-3 beta signaling, as well as invasiveness and expression of epithelial to mesenchymal transition markers in gastric cancer cell lines. CLEC2 prevents expression of PI3K subunits, in a SYK-dependent manner.

Abstract 867: Therapeutic potential of small molecule SYK inhibitors for treatment of primary B cell acute lymphoblastic leukemia

Abstract Background: B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Intensified and central nervous system (CNS)-directed chemotherapy has significantly improved outcomes for pediatric patients but are associated with late-effect morbidities. Moreover, ∼20% pediatric and a higher frequency of adult patients suffer relapses that are often fatal. Thus there is a need to develop therapies that target signaling abnormalities in B-ALL, which may reduce complications of CNS leukemia and decrease long-term morbidities. Rationale: Using a p53-/- SCID mouse model of B-ALL we observed pre-B cell receptor (pre-BCR)-independent activation of the spleen tyrosine kinase (SYK) and found that it was crucial for the proliferation and survival of these leukemias. We then asked whether abnormal SYK activation occurs in human B-ALL and whether these cells are sensitive to small molecule SYK inhibitors. Methods: Viably frozen diagnostic B-ALL samples from children (n=54) and adults (n=42) tested for sensitivity to SYK inhibitors R406 (Astra-Zeneca) and BAY61-3606 in a short-term in vitro proliferation assay. Phospho-flow cytometry was also performed to quantify phosphorylation of SYK and other signaling proteins in B-ALL samples. The R406 pro-drug (Fostamatinib: Fosta) was used in a xenotransplant assay to determine therapeutic potential of SYK inhibition in vivo. Results: Phospho-flow cytometry profiling of primary B-ALL samples revealed prominent phosphorylation of SYK (Y348) and downstream signaling proteins that was decreased by SYK inhibitors. Furthermore, SYK inhibitors significantly attenuated proliferation of pre-BCR-negative and pre-BCR-positive B-ALL samples indicating that SYK was required for their survival and proliferation. In contrast, FLT3 or SRC inhibitors did not inhibit proliferation of pediatric and adult B-ALL samples. Importantly, siRNA-mediated SYK knockdown also reduced proliferation of B-ALL cell lines. Therefore, we tested the therapeutic potential of SYK inhibition using xenotransplantion. NOD.SCID.gamma C-/- (NSG) mice were injected intrafemorally with primary B-ALL samples (n=9) and fed chow containing either vehicle (AIN-76A diet) or Fosta (AIN-76A diet with 2g Fosta/kg). Leukemia burden was assessed 4-8 weeks post-transplantation. Mice given the Fosta diet had significantly reduced numbers of leukemic blasts in their injected femurs, other bones, spleens and CNS as compared to vehicle-treated mice. In addition, Fosta treatment reduced spleen, liver and kidney weight in ALL-transplanted mice. Conclusion: SYK signaling is vital to B cell acute lymphoblastic leukemia survival; small molecule SYK inhibitors have therapeutic potential in poor-prognosis and relapsed B-ALL. 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 867. doi:1538-7445.AM2012-867

Activation of the protein kinase A pathway down regulates spleen tyrosine kinase in human T cells (63.30)

Abstract Cyclic adenosine monophosphate (cAMP)-mediated activation of the protein kinase A (PKA) pathway has an overall negative impact on T cell proliferation and down regulates or neutralizes signaling pathways of many genes. Spleen tyrosine kinase (SYK) is an important gene expressed differentially during T cell development and functions by phosphorylating downstream effector molecules in both T and B lymphocytes. We report here that PKA inhibitors promoted, whereas cAMP analogues suppressed SYK expression. Overexpression of a catalytic subunit of PKA significantly abrogated SYK protein and mRNA expression as well as the levels of phosphorylated SYK. A complete cAMP response element (CRE) site on SYK promoter was found functional and capable of binding CREMα and CREB as detected by EMSA and chromatin immunoprecipitation. CREMα overexpression reduced, while disruption of the CRE site enhanced the basal promoter activity of SYK. Collectively, the cAMP-mediated PKA pathway functions as the negative regulator of SYK expression by promoting direct binding of a repressor element to the SYK promoter.

Spleen Tyrosine Kinase Inhibition Prevents Chemokine- and Integrin-Mediated Stromal Protective Effects in Chronic Lymphocytic Leukemia.

Abstract 2356Poster Board II-333B cell chronic lymphocytic leukemia (CLL) remains incurable despite recent therapeutic advances, and identification of novel molecular targets in CLL is highly desirable. The microenvironment provides essential growth and survival signals to CLL cells and confers resistance to cytotoxic agents, a phenomenon termed cell adhesion-mediated drug resistance (CAM-DR). The chemokine CXCL12 and the integrin ligand VCAM have been identified as important microenvironmental pathways that mediate CLL cell survival and CAM-DR. We have previously demonstrated that inhibition of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, induces apoptosis in primary CLL cells when cultured alone or supported by stroma (Buchner et al., Cancer Res. 2009). In this study, we investigated SYK inhibition with regard to the interaction between CLL cells and the microenvironment.Stromal cell coculture resulted in SYK activation in primary CLL cells as indicated by phospho-protein analysis by flow cytometry (p<0.001) and immunoblotting. In vitro stimulation with 20 ng/ml recombinant CXCL12 or VLA-4 integrin ligation by contact to VCAM-1 precoated wells (10 μg/ml) for 15 sec had equivalent effects. SYK inhibition by the pharmacological SYK inhibitor R406 significantly reduced F-actin polymerization and Akt phosphorylation induced by CXCL12, integrin ligand, or stromal contact. In functional experiments, migration of CLL cells towards CXCL12 was reduced to 77% (p<0.001) by R406 in transwell culture. Likewise, CLL cell adhesion to VCAM was limited by SYK inhibition to 50% (p<0.01) compared to control cells in flow chamber experiments. In response to a number of physiological stimuli, including BCR ligation, integrins are activated through the modulation of their conformation to mediate high-affinity adhesion. Analysis of the expression of a high affinity epitope of CD29, the β1 chain of VLA-4, on CLL cells by flow cytometry with and without SYK inhibition revealed a significant decrease of 31% (p<0.0001), while the expression of total CD29 decreased only by 10% (p<0.01). To verify the specificity of the effects of pharmacological SYK inhibitors, we performed siRNA mediated silencing of SYK in primary CLL cells. 48 h after transfection, we observed a downregulation of SYK protein expression by flow cytometry by 27% (p<0.05), while the viability of the cells was not significantly affected for 72 h. Stimulation of control- and SYK-siRNA treated cells with VCAM significantly increased Akt phosphorylation in control but not in SYK-siRNA transfected cells. Furthermore, SYK-silenced CLL cells adhered significantly less to VCAM-coated surfaces than control-silenced CLL cells.Beyond inhibition of these defined pathways, R406 abrogated the protective effect of stromal coculture on CLL cell survival. The anti-apoptotic factor Mcl-1 was upregulated in CLL cells cultured with stroma compared to those cultured alone (mean MFI ± SEM: 144 ± 21 vs. 182 ± 23, p<0.001). SYK inhibition almost completely abolished the upregulation of Mcl-1 in stroma-cocultured CLL cells (mean MFI ± SEM: 182 ± 23 vs. 130 ± 15, p<0.001). Finally, SYK inhibition also prevented CAM-DR against fludarabine in stroma coculture.Taken together, our data demonstrate direct effects of SYK inhibition on chemokine and integrin-mediated CLL-stroma interactions and establish a novel therapeutic mechanism of action for SYK inhibitors in CLL. Indeed, a promising clinical activity in patients with relapsed/refractory CLL/SLL with an overall response rate of 54% has been reported for the oral prodrug of R406, fostamatinib disodium in CLL patients (Friedmann et al, ASH 2008). Of note, an unexplained transient but dramatic leukocyte flare during lymph node regression was reported in several of these patients. Our results offer a convincing explanation for this phenomenon and may even suggest that SYK blockade in CLL is a promising therapeutic principle not only due to inhibition of BCR signaling, but perhaps even more importantly by disrupting the contact of CLL cells with their protective microenvironment. Disclosures:No relevant conflicts of interest to declare.

Spleen Tyrosine Kinase Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

B-cell receptor signaling contributes to apoptosis resistance in chronic lymphocytic leukemia (CLL), limiting the efficacy of current therapeutic approaches. In this study, we investigated the expression of spleen tyrosine kinase (SYK), a key component of the B-cell receptor signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared with healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCgamma(2), signal transducers and activators of transcription 3, and extracellular signal regulated kinase 1/2 in CLL compared with healthy B cells, suggesting enhanced activation of these mediators in CLL. SYK inhibitors reduced phosphorylation of SYK downstream targets and induced apoptosis in primary CLL cells. With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70(+) cases. Cytotoxic effects of SYK inhibitors also associated with SYK protein expression, potentially predicting response to therapy. Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared with fludarabine therapy alone. We observed no stroma-contact-mediated drug resistance for SYK inhibitors as described for fludarabine treatment. CD40 ligation further enhanced efficacy of SYK inhibition. Our data provide mechanistic insight into the recently observed therapeutic effects of the SYK inhibitor R406 in CLL. Combination of SYK inhibitors with fludarabine might be a novel treatment option particularly for CLL patients with poor prognosis and should be further evaluated in clinical trials.

Structural Insights for Design of Potent Spleen Tyrosine Kinase Inhibitors from Crystallographic Analysis of Three Inhibitor Complexes

Spleen tyrosine kinase is considered an attractive drug target for the treatment of allergic and antibody mediated autoimmune diseases. We have determined the co-crystal structures of spleen tyrosine kinase complexed with three known inhibitors: YM193306, a 7-azaindole derivative and R406. The cis-cyclohexyldiamino moiety of YM193306 is forming four hydrophobically shielded polar interactions with the spleen tyrosine kinase protein and is therefore crucial for the high potency of this inhibitor. Its primary amino group is inducing a conformational change of the spleen tyrosine kinase DFG Asp side chain. The crystal structure of the 7-azaindole derivative bound to spleen tyrosine kinase is the first demonstration of a 2-substituted 7-azaindole bound to a protein kinase. Its indole-amide substituent is tightly packed between the N- and C-terminal kinase lobes. The co-crystal structure of the spleen tyrosine kinase-R406 complex shows two main differences to the previously reported structure of spleen tyrosine kinase soaked with R406: (i) the side chain of the highly conserved Lys is disordered and not forming a hydrogen bond to R406 and (ii) the DFG Asp side chain is pointing away from and not towards R406. The novel protein-ligand interactions and protein conformational changes revealed in these structures guide the rational design and structure-based optimization of second-generation spleen tyrosine kinase inhibitors.

Spleen Tyrosine Kinase (SYK) Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

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.

The prognostic value of Stathmin-1, S100A2, and SYK proteins in ER-positive primary breast cancer patients treated with adjuvant tamoxifen monotherapy: an immunohistochemical study

We recently found that DNA methylation of S100A2, spleen tyrosine kinase (SYK), and Stathmin-1 (STMN1) correlates with response to tamoxifen therapy in metastatic breast cancer. In this retrospective study, we investigated immunohistochemically whether these three markers are predictors of relapse in early breast cancer (EBC) patients treated with adjuvant tamoxifen alone. Immunohistochemical staining was performed for S100A2, SYK and STMN1 on a tissue microarray containing ER-positive invasive breast carcinomas from a study cohort of 215 operable breast cancer patients, who underwent radical local therapy and who were treated with adjuvant tamoxifen monotherapy. Cox regression was used to correlate staining intensity of the three markers with main endpoints in our study; disease-free survival (DFS), and disease-specific survival (DSS). In univariate analysis, only STMN1 staining intensity strongly correlated with DFS (P = 0.014) and DSS (P = 0.002). In the groups of low and high STMN1 intensity, DFS was 84% and 63%, and DSS was 89% and 70%. STMN1 retained its prognostic value for DFS (P = 0.002) and DSS (<0.001) in the multivariate model together with lymph node status. We found also a trend to better DFS in patients with low STMN1 intensity in both lymph node-positive (P = 0.001) and -negative patients (P = 0.065). As the tumour cells did not express S100A2 (except in one case) the potential prognostic value of this marker was not evaluated. Staining intensity of STMN1, but not SYK, predicted outcome in our collective of ER- positive tamoxifen treated EBC patients.