Lyn Expression Research Articles

Inhibition of SRC family kinases reduces myeloid-derived suppressor cells in head and neck cancer.

SRC family kinases (SFKs), a group of nonreceptor tyrosine kinases, modulate multiple cellular functions, such as cell proliferation, differentiation and metabolism. SFKs display aberrant activity in progressive stages of human cancers. However, the precise role of SFKs in the head and neck squamous cell carcinoma (HNSCC) signaling network is far from clear. In this study, we found that the inhibition of SFKs activity by dasatinib effectively reduced the tumor size and population of MDSCs in the HNSCC mouse model. Molecular analysis indicates that phosphorylation of LYN, rather than SRC, was inhibited by dasatinib treatment. Next, we analyzed LYN expression by immunostaining and found that it was overexpressed in the human HNSCC specimens. Moreover, LYN expression in stromal cells positively correlated with myeloid-derived suppressor cells (MDSCs) makers CD11b and CD33 in human HNSCC. The dual positive expression of LYN in epithelial and stromal cells (EPI+ SRT+ ) was associated with unfavorable overall survival of HNSCC patients. These findings indicate that SFKs may be a potential target for an effective immunotherapy of HNSCC by decreasing MDSCs and moreover, LYN will have an impact on such therapeutic strategy.

LYN expression predicts the response to dasatinib in a subpopulation of lung adenocarcinoma patients.

Therapies targeting SRC family kinases (SFKs) have shown efficacy in treating non-small cell lung cancer (NSCLC). However, recent clinical trials have found that the SFK inhibitor dasatinib is ineffective in some patient cohorts. Regardless, dasatinib treatment may benefit some NSCLC patient subgroups. Here, we investigated whether expression of LYN, a member of the SFK family, is associated with patient survival, the efficacy of dasatinib, and/or NSCLC cell viability. LYN expression was associated with poor overall survival in a multivariate analysis, and this association was strongest in non-smoker female patients with adenocarcinoma (ADC). In lung ADC cells, LYN expression enhanced cell proliferation, migration, and invasion. Dasatinib inhibited LYN activity and decreased cell viability in LYN-positive ADC cell lines and xenografts. Additionally, we identified the SFKs SRC and YES as candidate dasatinib targets in LYN-negative ADC cell lines. Our findings suggest that LYN is a useful prognostic marker and a selective target of dasatinib therapy in the lung ADC subpopulation especially in female non-smokers with lung ADC.

Tyrosine kinase LYN is an oncotarget in human cervical cancer: A quantitative proteomic based study.

Cervical cancer is one of the most common malignant tumor in women. The mechanisms of cervical cancer are intricate and have not been fully understood. Therefore, we employed iTRAQ to obtain novel proteins profile which participates in the tumor oncogenesis of cervical cancer. 3300 proteins were identified aberrantly expressed in cervical cancer, and western bolt was performed to validate the results of iTRAQ. Then, we selected LYN for further study. Immunohistochemistry identified that LYN expression was significantly increased in cervical cancer tissues than that in cancer adjacent normal cervical tissues and normal cervical tissues. The increased LYN expression was significantly correlated with cancer differentiation and FIGO stage. Silencing LYN inhibited cell proliferation, migration and invasion, conversely, overexpression LYN promoted cell proliferation, migration and invasion. In terms of mechanism, LYN could also promote cervical cancer cells metastasis through activating IL-6/STAT3 pathway. In vivo study, overexpression LYN promoted tumor growth, meanwhile knockdown LYN inhibited tumor growth. These results indicate that LYN tyrosine kinase is an oncogenic gene and can serve as a novel target for cervical cancer research and therapy.

Abstract 1682: Lyn drives cancer metastasis via post-translational regulation of SNAI proteins

Abstract Introduction: Metastasis is the most common cause of death from cancer and occurs when malignant cells discard epithelial restraints and acquire invasive abilities, facilitating their dissemination to permissive micro-environments. This process is enhanced by tumor cell activation of Epithelial Mesenchymal Transition (EMT), a (normally embryonic) developmental program in which epithelial cells assume a mesenchymal phenotype during gastrulation and organogenesis, allowing single cell invasive movement away from the ectodermal layer. Recent evidence strongly implicates EMT induction in malignant progression and treatment resistance. For example, EMT regulatory transcription factors are required for breast cancer metastasis. Several oncogenic pathways (growth factors, Src family, MAPK, AKT) induce EMT. Lyn tyrosine kinase, a member of Src family tyrosine kinase is up-regulated in advanced prostate cancer and has been reported to correlate with aggressive breast cancer. Our objective is to determine the role of Lyn tyrosine kinase in EMT. Methods: LNCaP (Lymph Node Metastasis of Prostate Cancer), BT-549 (Triple Negative Breast Cancer) and UM-UC-13 (Bladder Cancer) cells were transfected with Lyn siRNA; EMT markers were monitored by western blot and qRT-PCR and immunofluorescence, migration by scratch assay and invasion by Boyden chamber. Sub cellular localization of proteins was examined by IF and nuclear/cyto extraction. In vivo experiments were performed in UC13-luc cells with shRNA of Lyn. Results: Here we report that Lyn expression is low in epithelial cells and is up-regulated in mesenchymal cells. Targeting Lyn using siRNA decreases EMT markers (Fibronectin, Vimentin and Zeb-1) at both mRNA and protein levels while increasing the epithelial marker (E-cadherin). Moreover, we found that Lyn siRNA decreases cell migration and invasion. Thisis decrease in mesenchymal phenotype can be attributed to the decrease in the amount of Slug and Snail, transcriptional repressors of E-Cadherin and activator of Vimentin and Fibronectin. Interestingly, we found that Lyn knockout induces a decrease of SLUG only at protein levels and not at mRNA levels. We discovered that Lyn triggers a signaling cascade through Vav-Rac-Pak1 pathway to alter sub cellular localization of the SNAI proteins leading to their proteasomal degradation. This effect results in decreased invasion and migration in vitro as well as decreased metastasis in vivo. Conclusion: Expression of Lyn kinase can be correlated to low prognosis and aggressive/metastatic phenotype. We show that knocking down Lyn by siRNA initiates a switch to a more epithelial phenotype reducing cell migration and invasion. Citation Format: Daksh Thaper, Sepideh Vahid, Ka Mun Nip, Igor Moskalev, Sebastian Frees, Morgan E. Roberts, Krisi Ketola, Kenneth W. Harder, Jennifer L. Bishop, Amina Zoubeidi. Lyn drives cancer metastasis via post-translational regulation of SNAI proteins. [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 1682.

Hydroquinone-induced FOXP3-ADAM17-Lyn-Akt-p21 signaling axis promotes malignant progression of human leukemia U937 cells

Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5μM HQ for 24h, and the cells were re-suspended in serum-containing medium without HQ for 2days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5μM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced β-TrCP mRNA stability and suppressed β-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.

Deregulated Expression of SRC, LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis.

Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced SRC and LYN methylation and increased CKB methylation was associated with gastric cancer. The reduced SRC and LYN methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced CKB methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies.

N-3 Polyunsaturated fatty acids inhibit Fc ε receptor I-mediated mast cell activation

In vivo models show that n-3 polyunsaturated fatty acids (PUFA) inhibit some of the processes associated with allergic inflammation but the direct effect of n-3 PUFA on mast cells, the major effector cells in allergy, is poorly understood. We sought to determine the effect and mechanism of n-3 PUFA on Fc ε receptor I (FcεRI)-mediated signal transduction and mast cell activation. Bone marrow-derived mast cells (BMMC) were differentiated from bone marrow obtained from C57BL/6 wild-type (WT) and fat-1 transgenic mice. The fat-1 mice express fatty acid n-3 desaturase and produce endogenous n-3 PUFA. For comparison, exogenous n-3 PUFA were supplemented to WT BMMC and human mast cell (LAD2) cultures. Fat-1 BMMC released less β-hexosaminidase (β-hex) and cysteinyl leukotrienes and produced less tumor necrosis factor and chemokine (C-C motif) ligand 2. n-3 PUFA supplementation reduced LAD2 and BMMC degranulation (β-hex release) following FcεRI activation. Fat-1 BMMC expressed less constitutive Lyn and linker of activated T cells (LAT), and FcεRI-mediated phosphorylation of Lyn, spleen tyrosine kinase and LAT were reduced in fat-1 BMMC. Although the expression of surface and whole cell FcεRI was similar in WT and fat-1 BMMC, unstimulated fat-1 BMMC showed reduced FcεRI localization to lipid rafts, and stimulation with antigen resulted in aberrant FcεRI shuttling to the rafts. Our results show that n-3 PUFA suppress FcεRI-mediated activation of mast cells, which results in reduced mediator release. This effect is associated with a decrease in LAT and Lyn expression as well as abnormal shuttling of FcεRI to lipid rafts.

Abstract 4106: Lyn kinase promotes metastasis through EMT in cancers

Abstract Introduction: Metastasis is the most common cause of death from cancer and occurs when malignant cells discard epithelial restraints and acquire invasive abilities, facilitating their dissemination to permissive micro-environments. This process is enhanced by tumor cell activation of Epithelial Mesenchymal Transition (EMT), a (normally embryonic) developmental program in which epithelial cells assume a mesenchymal phenotype during gastrulation and organogenesis, allowing single cell invasive movement away from the ectodermal layer. Recent evidence strongly implicates EMT induction in malignant progression and treatment resistance. For example, EMT regulatory transcription factors are required for breast cancer metastasis. Several oncogenic pathways (growth factors, Src family, MAPK, AKT) induce EMT. Lyn tyrosine kinase, a member of Src family tyrosine kinase is up-regulated in advanced prostate cancer and has been reported to correlate with aggressive breast cancer. Our objective is to determine the role of Lyn tyrosine kinase in EMT. Methods: LNCaP, BT-549, UC-13 cells were transfected with Lyn siRNA; EMT markers were monitored by western blot and qRT-PCR and immunofluorescence, migration by scratch assay, invasion by Boyden chamber and matrix metalloproteinase (MMP) activity by Zymography. Lyn specific Kinase inhibitor Bafetinib will also be used along with over-expression experiments with Lyn Kinase Dead and Constitutively active mutants. Results: Here we report that Lyn expression is low in epithelial cells and is up-regulated in mesenchymal cells. Targeting Lyn using siRNA decreases EMT markers (Fibronectin, Vimentin) at both mRNA and protein levels while increasing the epithelial marker (E-cadherin). Moreover, we found that targeting Lyn by siRNA/shRNA and small molecule inhibitor Bafetinib decreases cell migration, invasion and the activity of MMPs. This decrease in mesenchymal phenotype can be attributed to the decrease in the amount of Slug and Snail, transcriptional repressors of E-Cadherin and activators of Vimentin. Consequently, we also found that over-expressing Lyn in both as WT or constitutively active (CA) mutant rescues expression of Slug and Snail, inducing EMT, increased cell migration and invasion while Kinase Dead (KD) mutant has no effect. Conclusion: Expression of Lyn kinase can be correlated to low prognosis and aggressive/metastatic phenotype. We show that targeting Lyn activity initiates a switch to a more epithelial phenotype reducing cell migration and invasion. Impact: The data suggests that Lyn tyrosine kinase plays a role in Epithelial Mesenchymal Transition and could be considered as a target for metastatic disease; especially in the more aggressive forms of cancer like Triple Negative Breast Cancer or Castration Resistant Prostate Cancer. This could be a realistic therapeutic option, as the Lyn small molecule inhibitor Bafetinib, is currently in clinical trials for treatment of several cancers. Note: This abstract was not presented at the meeting. Citation Format: Daksh Thaper, Sepideh Vahid, Ka Mun Nip, Kirsi Ketola, Jennifer Bishop, Amina Zoubeidi. Lyn kinase promotes metastasis through EMT in cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4106. doi:10.1158/1538-7445.AM2015-4106

CBL controls a tyrosine kinase network involving AXL, SYK and LYN in nilotinib-resistant chronic myeloid leukaemia.

A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells.

Mechanism of expression of the Src family kinases (Fgr, Hck, Lyn) and SSeCKS in non-alcoholic steatohepatitis

To investigate the role of Src family kinases (Fgr, Hck, Lyn) and the major protein kinase C substrate SSeCKS in non-alcoholic steatohepatitis (NASH) and determine the possible mechanism regulating differential expression. Kupffer cells were stimulated with CCL4 and effect on SSeCKS, Hck, Fgr, and Lyn expression was detected by real-time reverse transcription-PCR. Male Sprague-Dawley rats were used to create a NASH model by feeing a high fat diet. The modeled rats were divided into a model group and a normal group. After sacrifice, the extent of hepatic steatosis and inflammation was assessed, and the expression levels of SSeCKS and Hck, Fgr, Lyn were detected by immunohistochemical staining. Expression of Lyn and Hck was decreased in the CCL4-stimulated Kupffer cells and the change in expression level was positively associated with levels of inflammatory stimuli (P < 0.01). The change in expression of SSeCKS in the CCL4-stimulated Kupffer cells was negatively correlated with inflammatory stimuli (P < 0.01). Fgr expression was very low in the unstimulated Kupffer cells and was not affected by the exposure to inflammatory stimuli. The number of inflammatory cells in the liver tissues of rars were negatively correlated with expression of Lyn, Hck and SSeCKS (P < 0.01), with low negative correlation for Lyn (r =-0.398, P < 0.01) and moderate negative correlation for Hck (r=-0.508, P < 0.01); the Lyn and Hck expression levels were highly positively correlated (r =0.942, P < 0.01). Src family kinases (Lyn, Hck and Fgr) and SSeCKS are involved in development and progression of NASH, and their differential expression patterns are associated to a certain extent. The factors may represent potential targets of therapy for NASH-related inflammation.

Immunoglobulin Heavy Chain (IgH) Knockout Inhibits Proliferation of Pre-BCR+ B-Cell Acute Lymphoblastic Leukemia (B-ALL) Via a FOXO1 and MYC Dependent Mechanism

A pivotal step during B-cell development is the expression of the precursor B-cell receptor (pre-BCR) by pre-B lymphocytes (cyto-Igµ+, surface-IgM-). The pre-BCR represents an immature form of the BCR and consists of two immunoglobulin heavy chains (IgH), two surrogate light chains (SLC) and the signal transducing adapter proteins Igα and Igβ. A functional pre-BCR drives proliferation of pre-B-cells, ensuring their further differentiation into mature B-cells. By immunophenotype, ~20% of B-cell acute lymphoblastic leukemia (B-ALL) cases originate from the pre-B-cell stage (pre-B-ALL) of lymphocyte development and might therefore also express the pre-BCR. In view of the importance of pre-BCR signaling for normal pre-B-cell development, we hypothesize that it is exploited by pre-B-ALL for malignant growth and proliferation.A hallmark of active pre-BCR signaling is the continuous internalization of pre-BCRs, resulting in low pre-BCR surface expression. Using this phenotype of active pre-BCR signaling (low pre-BCR expression and high phosphorylation of the pre-BCR associated kinases LYN and SYK), we identified pre-BCR+ ALL cell lines (RCH-ACV, SMS-SB and Nalm6) and xenograft expanded patient samples. To study the role of the pre-BCR in these cells, we rendered RCH-ACV and SMS-SB pre-BCR null by using CRISPR/CAS9 gene editing with guide RNAs specific for the hypervariable region (recombined V, D, and J segments) of their expressed IgH allele. As identified by flow cytometry for the pre-BCR, deficient RCH-ACV and SMS-SB cells exhibited reduced viability and impaired proliferation when compared to their pre-BCR+ controls (Figure 1). Pre-BCR- cells showed reduced baseline phosphorylation of CD19, VAV1 and AKT. Interestingly, BTK and ERK phosphorylation were not affected. These results provide evidence for the dependency of pre-BCR+ ALL on pre-BCR signaling and suggest selective involvement of the PI3K-AKT pathway.We also investigated the effects of pharmacological pre-BCR inhibition by treating pre-BCR+ and pre-BCR- ALL cell lines and xenograft expanded primary patient samples with PRT318, a small-molecule inhibitor of spleen tyrosine kinase (SYK). In pre-BCR+ ALL PRT318 blocked cell proliferation and selectively inhibited AKT phosphorylation, thus mimicking the effects of IgH knockout. Pre-BCR- ALL cells were resistant to PRT318.Key effectors of the pre-BCR during normal B-cell development are FOXO transcription factors. In line with this, we found reduced FOXO1 phosphorylation and increased FOXO1 total protein levels after IgH knockout as well as after treatment with PRT318. This was accompanied by an increase in the FOXO1 transcriptional targets p27 and BLNK, suggesting increased FOXO1 transcriptional activity in response to the inhibition of pre-BCR signaling. To study the contribution of FOXO1 to the effects of IgH knockout and SYK inhibition more thoroughly, we expressed constitutively active FOXO1 (FOXO1-3A) in the pre-BCR+ ALL cell line RCH-ACV and consequently assessed its effects on cell proliferation and protein expression. Similar to IgH knockout and PRT318, FOXO1-3A reduced cell proliferation and increased p27 and BLNK protein levels, confirming FOXO1 as an important downstream target of pre-BCR signaling in B-ALL.To identify additional effectors of the pre-BCR in B-ALL we performed gene expression profiling (GEP) to compare pre-BCR+ and pre-BCR- cells of RCH-ACV and SMS-SB. Gene set enrichment analysis (GSEA) showed that IgH knockout resulted in significant enrichment for gene sets associated with down-modulation of MYC activity. This was confirmed by Western blot analysis of MYC total protein levels, and consistent with the finding of reduced MYC protein in PRT318-treated and FOXO1-3A-expressing pre-BCR+ cells, all indicating that pre-BCR signaling modulates MYC activity through a mechanism involving SYK and FOXO1.In conclusion, we provide evidence for the dependence of certain B-ALL subgroups on pre-BCR signaling. According to our data this is mainly due to pre-BCR-induced inactivation of FOXO1 and the subsequent deregulation of MYC. Importantly, pharmacological inhibition of pre-BCR signaling with the SYK inhibitor PRT318 completely reversed these effects, therefore providing a rationale for the use of SYK inhibitors in pre-BCR+ subgroups of B-ALL.Figure 1: [Display omitted] DisclosuresCoffey:Portola Pharmaceuticals: Employment, Equity Ownership.

A Novel Path for ATRA Differentiation Therapy in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

Acute myeloid leukemia (AML) is characterized by accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, among which IDH mutations occur in 15% of AML patients. These mutations lead to a block in erythroid commitment while they may also bias hematopoietic differentiation to myeloid lineage. Interestingly, Lyn tyrosine kinase is required for erythroid differentiation and we have observed a reduction of Lyn expression in the presence of IDH1-R132H mutation. It is also a negative regulator of ATRA-induced granulocytic differentiation. Accordingly, we hypothesized that IDH mutations may sensitize AML cells to ATRA-induced differentiation.Here, we report that clinically achievable doses of ATRA are sufficient to trigger differentiation specifically on AML cell lines, primary patient samples and xenograft mice models carrying IDH1 mutation as observed by an increase in CD11b expression, granulocytic enzyme activity and morphologic changes in May-Grunwald-Giemsa staining. We also showed that ATRA-induced terminal granulocytic differentiation increases apoptosis while decreases proliferation and colony formation specifically in IDH1 mutant cells. Moreover, inhibition of IDH1-R132H activity reduced ATRA-sensitivity while increasing expression of IDH mutation correlated with highest ATRA sensitivity. Furthermore, treatment with a cell-permeable form of the oncometabolite specifically produced by the mutant (eg. 2-HydroxyGlutarate) sensitized AML cells to ATRA-induced differentiation. Finally, because ATRA-induced differentiation triggers a transient increase of Lyn activation, its association with Lyn inhibitors synergistically increased ATRA-induced differentiation of IDH mutant blasts.In summary, our results showed that IDH mutations by producing 2-HG sensitized leukemic blasts to ATRA and that this synergizes with Lyn inhibition. Since 2HG concentration reaches millimolar in AML patient serum and is 100-fold higher in IDH mutated patients than in non-mutated ones, we would predict a strong efficacy and specificity of ATRA. Furthermore, as IDH mutations are systematically conserved at relapse, this therapeutic strategy might be promising to achieve a long-term remission specifically for this AML patient subgroup. DisclosuresNo relevant conflicts of interest to declare.

Differential expression of Toll-like receptor (TLR) and B cell receptor (BCR) signaling molecules in primary diffuse large B-cell lymphoma of the central nervous system.

Primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) is a distinct and aggressive lymphoma that is confined to CNS. Since, central nervous system is barrier-protected and immunologically silent; role of TLR/BCR signaling in pathogenesis and biology of CNS DLBCL is intriguing. Genomic mutations in key regulators of TLR/BCR signaling pathway (MYD88/CD79B/CARD11) have recently been reported in this disease. These observations raised possible implications in novel targeted therapies; however, expression pattern of molecules related to TLR/BCR pathways in this lymphoma remains unknown. We have analyzed the expression of 19 genes encoding TLR/BCR pathways and targets in CNS DLBCLs (n = 20) by Nanostring nCounter™ analysis and compared it with expression patterns in purified reactive B-lymphocytes and systemic diffuse large B cell lymphoma (DLBCL) (n = 20). Relative expression of TLR4, TLR5, TLR9, CD79B and BLNK was higher in CNS DLBCLs than in control B-lymphocytes; where as TLR7, MALT1, BCL10, CD79A and LYN was lower in CNS DLBCLs (P < 0.0001). When compared with systemic DLBCL samples, higher expression of TLR9, CD79B, CARD11, LYN and BLNK was noted in CNS DLBCL (>1.5 fold change; P < 0.01). The B cell receptor molecules like BLNK and CD79B were also associated with higher expression of MYD88 dependent TLRs (TLR4/5/9). In conclusion, we have shown over expression of TLR/BCR related genes or their targets, where genomic mutations have commonly been identified in CNS DLBCL. We have also demonstrated that TLR over expression closely relate with up regulation of genes associated with BCR pathway like CD79B/BLNK and CARD11, which play an important role in NF-kB pathway activation. Our results provide an important insight into the possibility of TLR and/or B-cell receptor signaling molecules as possible therapeutic targets in CNS DLBCL.

Abstract 2785: LYN is a new prognostic and therapeutic target in non-small cell lung cancer

Abstract SRC family kinases (SFKs) have a strong rationale as a clinical strategy in non-small cell lung cancer (NSCLC). Unfortunately, the reviewed study showed disappointing activity of SFK inhibitor dasatinib in molecularly unselected patient cohort. Therefore, the studies in NSCLC should be investigated to identify patients more likely to benefit from dasatinib either as monotherapy or in combination with other agents. Here, we considered that LYN may play specific roles in lung cancer and therefore analysed survival prognosis according to the expression of LYN and the effect of dasatinib on the inhibition of cell growth and LYN activity. We evaluated LYN expression by immunohistochemistry on 446 NSCLCs using paraffin-embedded tissues. Elevated LYN expression level of NSCLCs, especially in adenocarcinoma, female, and non-smoker, affected poor overall survival (OS, P=0.036). In lung adenocarcinoma cell lines, LYN expression impacted on cell proliferation, migration and invasion. Dasatinib treatment resulted in decreased cell viability in most of these cell lines. Also, we found that dasatinib suppressed active tyrosine phosphorylation of LYN. In the resistant cell lines, H23 and SK-LU-1, which showed elevated epidermal growth factor (EGFR) and MET activities, combined treatment of EGFR or MET inhibitor with dasatinib revealed the synergistic effects on decreased cell viability. Our findings highly suggest that LYN has potential as a new selected target molecule of SFKs in the treatment of NSCLCs. Citation Format: Yu Jin Kim, Minjung Sung, Min Jeong Park, Young Kee Shin, Yoon-La Choi. LYN is a new prognostic and therapeutic target in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2785. doi:10.1158/1538-7445.AM2014-2785

Macrophage migration inhibitory factor down-regulates the RANKL-RANK signaling pathway by activating Lyn tyrosine kinase in mouse models.

Macrophage migration inhibitory factor (MIF) is an important modulator of innate and adaptive immunity as well as local inflammatory responses. We previously reported that MIF down-regulated osteoclastogenesis through a mechanism that requires CD74. The aim of the current study was to examine whether MIF modulates osteoclastogenesis through Lyn phosphorylation, and whether down-regulation of RANKL-mediated signaling requires the association of CD74, CD44, and Lyn. CD74-knockout (CD74-KO), CD44-KO, and Lyn-KO mouse models were used to investigate whether Lyn requires these receptors and coreceptors. The effects of MIF on osteoclastogenesis were assessed using Western blot analysis, small interfering RNA (siRNA)-targeted down-regulation of Lyn, Lyn-KO mice, and real-time imaging of Lyn molecules to surface proteins. MIF treatment induced Lyn expression, and MIF down-regulated RANKL-induced activator protein 1 (AP-1) and the Syk/phospholipase Cγ cascade during osteoclastogenesis through activated Lyn tyrosine kinase. The results of immunoprecipitation studies revealed that MIF receptors associated with Lyn in response to MIF treatment. Studies using Lyn-specific siRNA and Lyn-KO mice confirmed our findings. Our findings indicate that the tyrosine kinase Lyn is activated when MIF binds to its receptor CD74 and its coreceptor CD44 and, in turn, down-regulates the RANKL-mediated signaling cascade by suppressing NF-ATc1 protein expression through down-regulation of AP-1 and calcium signaling components.

PSTPIP2 dysregulation contributes to aberrant terminal differentiation in GATA-1-deficient megakaryocytes by activating LYN

GATA1 mutations are tightly associated with transient myeloproliferative disorder (TMD) and acute megakaryoblstic leukemia (AMKL) in children with Down syndrome. Numerous genes are altered in GATA-1-deficient megakaryocytes, which may contribute to the hyperproliferation and abnormal terminal differentiation of these malignant cells. In this study, we demonstrate that Pstpip2 is a GATA-1-repressed gene that controls megakaryopoiesis. Ectopic expression of PSTPIP2 impaired megakaryocytic differentiation as evidenced by a decrease of CD41 expression and reduced DNA content in K562 cells. PSTPIP2 overexpression also caused enhanced activation of Src family kinases and subsequently reduced ERK phosphorylation. Consistently, PSTPIP2 knockdown showed the opposite effect on differentiation and signaling. Moreover, the W232A mutant of PSTPIP2, defective in its interaction with PEST family phosphatases that recruit c-Src terminal kinase (CSK) to suppress Src family kinases, failed to inhibit differentiation and lost its ability to enhance Src family kinases or reduce ERK phosphorylation. In fact, the W232A mutant of PSTPIP2 promoted megakaryocyte differentiation. These observations suggest that PSTPIP2 recruiting PEST phosphatases somehow blocked CSK activity and led to enhanced activation of Src family kinases and reduced ERK phosphorylation, which ultimately repressed megakaryocyte differentiation. Supporting this idea, PSTPIP2 interacted with LYN and the expression of a dominant negative LYN (LYN DN) overwhelmed the inhibitory effect of PSTPIP2 on differentiation and ERK signaling. In addition, a constitutively active LYN (LYN CA) normalized the enhanced megakaryocyte differentiation and repressed ERK signaling in PSTPIP2 knockdown cells. Finally, we found that PSTPIP2 repressed ERK signaling, differentiation, and proliferation and verified that PSTPIP2 upregulation repressed megakaryocyte development in primary mouse bone marrow cells. Our study thus reveals a novel mechanism by which dysregulation of PSTPIP2 due to GATA-1 deficiency may contribute to abnormal megakaryocyte proliferation and differentiation in pathogenesis of related diseases.

In Chronic Lymphocytic Leukemia PD-1 Is Expressed Independently From PDCD1 Gene Polymorphisms and Does Not Influence BCR Signaling

BackgroundThe programmed death-1 (PD-1, CD279) is a negative lymphocytic immunoreceptor responsible for peripheral tolerance. Through inhibition of TCR/BCR signaling PD-1 impairs activation and effectors functions of T and B lymphocytes, where it is expressed upon activation. PD-1 was described on exhausted T cells and it is aberrantly expressed in several malignancies, where a PD-1 pathway was related to a potential tumor escape mechanism from the immunosurveillance. Previously, we characterized PD-1 as a novel phenotypic feature of chronic lymphocytic leukemia (CLL) cells. PD-1 was up-regulated on both transcript and surface protein levels in CLL patients in comparison to healthy volunteers (HVs). Moreover, in vitro activated CLL cells in conditions mimicking microenvironment of proliferation centres up-regulated PD-1 on their surface. AimsDespite aberrant expression in CLL patients, PD-1 mechanism and function in CLL is unclear. To investigate PD-1 influence on BCR signaling in CLL cells we analyzed expression and phosphorylation of SYK, LYN, and ZAP-70 tyrosine kinases. To verify a relation of PD-1 expression to microenvironment in vivo, we analyzed PD-1 expression in mononuclears from 149 peripheral blood (PBMC) and 140 bone marrow (BMMC) CLL samples, representing accumulation and proliferation disease compartment respectively and 30 healthy PBMC samples. Finally, we characterized polymorphism of PDCD1 gene in 114 CLL patients and 150 HVs. MethodsQuantitative reverse transcriptase PCR was performed for the PD-1 transcript assessment in 182 CLL patients (148 PBMC, 140 BMMC). Expression and phosphorylation of tyrosine kinases were analyzed with western blot (for SYK and LYN) and flow cytometric methods (for ZAP-70 and PD-1) in CD19+ magnetically separated cells from 12 CLL patients. For each kinase two separate phosphorylation sites were analyzed, responsible either for activation or inhibition respectively: Y525/526 and Y323 for SYK, Y507 and Y396 for LYN, Y315/319 and Y292 for ZAP-70. Five single nucleotide polymorphism sites (SNPs) were analyzed by restriction fragments length polymorphism (RFLP-PCR) in group of 114 CLL patients and 150 HVs. ResultsThe median level of PD-1 transcripts in CLL patients was higher in comparison with HVs (p<0.0001), but there was no difference between blood and bone marrow compartments. The expression and phosphorylation of LYN and SYK were not dependent on PD-1 expression in 12 CLL patients, with PD-1 range from 4.2%. to 67.53%. Flow cytometric analysis showed that ZAP-70 expression was not related to the PD-1 level. Median percentages of cells with phosphorylated ZAP-70 activation/inhibition sites were low (Y315/319: 0.62%, Y292: 0.5%). Phosphorylation of Y292 showed strong negative correlation with PD-1 level (r2=-0.8, p=0.0019). Analysis of PDCD1 polymorphism showed that neither of SNPs frequency (loci: rs36084323, rs11568821, rs2227981, rs2227982, rs41386349) was different in CLL group compared to HVs. Particular substitutions were not related to the PD-1 expression level. Frequencies of haplotypes in CLL and HVs showed no significant difference. Linkage disequilibrium comparison of CLL and HVs revealed complete dependency of rs2227981/rs2227982 (p=0.03) and rs2227981/rs36084323 (p=0.03) loci in CLL patients. Summary / ConclusionPD-1 expression is strongly up-regulated in CLL patients regardless microenvironment of leukemic cells. No PD-1 influence on expression and activation of BCR related kinases might suggest impaired or alternative function of PD-1 pathway in CLL cells. Characterization of PDCD1 polymorphism revealed no association of PD-1 SNPs with CLL, therefore, PD-1 up-regulation might not be driven by genetic alternations. Disclosures:No relevant conflicts of interest to declare.

Expression of LYN and PTEN genes in chronic myeloid leukemia and their importance in therapeutic strategy

Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are the current treatment of chronic myeloid leukemia (CML). BCR-ABL1 point mutations are the principal cause of resistance to treatment; however other mechanisms could be involved in failure to TKI therapy. LYN is a src kinase protein that regulates survival and responsiveness of tumor cells by a BCR-ABL1 independent mechanism. PTEN tumor suppressor gene is downregulated by BCR-ABL1 in CML stem cells and its deletion is associated with acceleration of disease. In this study we evaluated the expression of LYN, PTEN and the ratio of both genes in 40 healthy donors (HD) and in 139 CML patients; 88 of them resistant to TKI in different phases of disease and 51 in chronic phase classified as optimal responders (OR) to TKI [40 treated with imatinib or nilotinib (OR-IN) and 11 treated with dasatinib (OR-D) therapy]. When we analyzed the gene expression values of LYN, an increase was observed only in advanced stages of the disease, however, when we analyzed the ratio between LYN and PTEN genes, the group of resistant patients in chronic phase in imatinib or nilotinib treatment (CP-IN) also showed a significant increase. Resistant patients treated with dasatinib, a src kinase inhibitor, presented a similar ratio to the observed in HD. In addition, the LYN/PTEN ratio and the LYN expression showed a direct significant correlation with BCR-ABL1 transcript levels in unmutated resistant patients treated with non-src kinase inhibitors. We were able to identify 8/35 (23%) of cases in CP-IN and 4/12 (33%) in accelerated phase and blast phase (AP/BC-IN), in which resistance could be associated with an increase in the ratio of the LYN/PTEN. Our data suggest that the LYN/PTEN expression ratio may be a sensitive monitor of disease progression in unmutated CML patients under imatinib or nilotinib treatment. This ratio could detect cases when resistance is related to altered LYN expression, suggesting that the treatment change to a src kinase inhibitor would be most suitable to overcome resistance.

Lyn, a Src family kinase, regulates activation of epidermal growth factor receptors in lung adenocarcinoma cells

BackgroundActivation of receptors for growth factors on lung epithelial cells is essential for transformation into tumor cells, supporting their viability and proliferation. In most lung cancer patients, EGFR is constitutively activated without evidence of mutation. Defining mechanisms for constitutive activation of EGFR could elucidate additional targets for therapy of lung cancers.MethodsThe approach was to identify lung cancer cell lines with constitutively activated EGFR and use systematic selection of inhibitors to evaluate their effects on specific EGFR phosphorylations and downstream signaling pathways. Interactions between receptors, kinases, and scaffolding proteins were investigated by co-immunoprecipitation plus Western blotting.ResultsThe results revealed a dependence on Src family of tyrosine kinases for downstream signaling and cell growth. Lyn, a Src family kinase functional in normal and malignant B-lymphocytes, was a defining signal transducer required for EGFR signaling in Calu3 cell line. Src family kinase activation in turn, was dependent on PKCßII. Lyn and PKC exist in membrane complexes of RACK1 and in association with EGFR which pairs with other receptor partners. Silencing of Lyn expression with interfering siRNA decreased EGFR activation and cell viability.ConclusionsThe importance of Src family kinases and PKCßII in the initiation of the EGFR signaling pathway in lung tumor cells was demonstrated. We conclude that phosphorylation of EGFR is mediated through PKCßII regulation of Lyn activation, and occurs in association with RACK1 and Cbp/PAG proteins. We suggest that protein complexes in cell membranes, including lipid rafts, may serve as novel targets for combination therapies with EGFR and Src Family Kinase inhibitors in lung cancer.

Genetic deletion of CD36 enhances injury after acute neonatal stroke

The scavenger receptor CD36 is injurious in acute experimental focal stroke and neurodegenerative diseases in the adult. We investigated the effects of genetic deletion of CD36 (CD36ko) on acute injury, and oxidative and inflammatory signaling after neonatal stroke. Postnatal day 9 CD36ko and wild-type (WT) mice were subjected to a transient middle cerebral artery occlusion (MCAO). Injury, phagocytosis of dying cells, and CD36 inflammatory signaling were determined. While the volume of tissue at risk by diffusion-weighted magnetic resonance imaging during MCAO was similar in neonatal CD36ko and WT mice, by 24 hours after reperfusion, injury was more severe in CD36ko and was associated with increased caspase-3 cleavage and reduced engulfment of neurons expressing cleaved caspase-3 by activated microglia. No significant superoxide generation was observed in activated microglia in injured WT, whereas increased superoxide production in vessels and nuclear factor (NF)-κB activation induced by MCAO were unaffected by lack of CD36. Lyn expression was higher in injured CD36ko, and cell type-specific patterns of Lyn expression were altered; Lyn was expressed in endothelial cells and microglia in WT but predominantly in dying neurons in CD36ko. Lack of CD36 results in poorer short-term outcome from neonatal focal stroke due to lack of attenuation of NF-κB-mediated inflammation and diminished removal of apoptotic neuronal debris. Although inhibition of CD36 does not seem to be a good therapeutic target for protection after acute neonatal stroke, as it is after adult stroke, seeking better understanding of CD36 signaling in particular cell populations may reveal important therapeutic targets for neonatal stroke.