Lyn mRNA Research Articles

Nanoformulation of dasatinib cannot overcome therapy resistance of pancreatic cancer cells with low LYN kinase expression

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most difficult to treat tumors. The Src (sarcoma) inhibitor dasatinib (DASA) has shown promising efficacy in preclinical studies of PDAC. However, clinical confirmation could not be achieved. Overall, our aim was to deliver arguments for the possible reinitiating clinical testing of this compound in a biomarker-stratifying therapy trial for PDAC patients. We tested if the nanofunctionalization of DASA can increase the drug efficacy and whether certain Src members can function as clinical predictive biomarkers.MethodsMethods include manufacturing of poly(vinyl alcohol) stabilized gold nanoparticles and their drug loading, dynamic light scattering, transmission electron microscopy, thermogravimetric analysis, Zeta potential measurement, sterile human cell culture, cell growth quantification, accessing and evaluating transcriptome and clinical data from molecular tumor dataset TCGA, as well as various statistical analyses.ResultsWe generated homo-dispersed nanofunctionalized DASA as an AuNP@PVA-DASA conjugate. The composite did not enhance the anti-growth effect of DASA on PDAC cell lines. The cell model with high LYN expression showed the strongest response to the therapy. We confirm deregulated Src kinetome activity as a prevalent feature of PDAC by revealing mRNA levels associated with higher malignancy grade of tumors. BLK (B lymphocyte kinase) expression predicts shorter overall survival of diabetic PDAC patients.ConclusionsNanofunctionalization of DASA needs further improvement to overcome the therapy resistance of PDAC. LYN mRNA is augmented in tumors with higher malignancy and can serve as a predictive biomarker for the therapy resistance of PDAC cells against DASA. Studying the biological roles of BLK might help to identify underlying molecular mechanisms associated with PDAC in diabetic patients.

In Vitro Validation of the Therapeutic Potential of Dendrimer-Based Nanoformulations against Tumor Stem Cells

Tumor cells with stem cell properties are considered to play major roles in promoting the development and malignant behavior of aggressive cancers. Therapeutic strategies that efficiently eradicate such tumor stem cells are of highest clinical need. Herein, we performed the validation of the polycationic phosphorus dendrimer-based approach for small interfering RNAs delivery in in vitro stem-like cells as models. As a therapeutic target, we chose Lyn, a member of the Src family kinases as an example of a prominent enzyme class widely discussed as a potent anti-cancer intervention point. Our selection is guided by our discovery that Lyn mRNA expression level in glioma, a class of brain tumors, possesses significant negative clinical predictive value, promoting its potential as a therapeutic target for future molecular-targeted treatments. We then showed that anti-Lyn siRNA, delivered into Lyn-expressing glioma cell model reduces the cell viability, a fact that was not observed in a cell model that lacks Lyn-expression. Furthermore, we have found that the dendrimer itself influences various parameters of the cells such as the expression of surface markers PD-L1, TIM-3 and CD47, targets for immune recognition and other biological processes suggested to be regulating glioblastoma cell invasion. Our findings prove the potential of dendrimer-based platforms for therapeutic applications, which might help to eradicate the population of cancer cells with augmented chemotherapy resistance. Moreover, the results further promote our functional stem cell technology as suitable component in early stage drug development.

Expression of the Src-family kinase LynA tunes myeloid-cell signaling and function

Abstract The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) upon receptor ligation, is a critical factor regulating myeloid cell activation. The SFK Lyn has roles in activating myeloid cells, by phosphorylating ITAMs, and also in inducing suppressive signaling via phosphatase activation. Alternative splicing of lyn mRNA produces two proteins, LynA and LynB, and emerging evidence from our lab and others suggests that LynA and LynB interact with different proteins and preferentially activate different signal cascades. In macrophages LynA is uniquely susceptible to rapid ubiquitin-mediated degradation and functions as a rheostat that regulates ITAM signal strength. Phosphorylation of tyrosine 32 in the unique region of LynA preferentially targets LynA for polyubiquitination by the E3 ligase c-Cbl. In contrast to macrophages, mast cells express little c-Cbl. Upon activation, LynA is not rapidly degraded in mast cells, and SFK-mediated signaling is amplified relative to macrophages. Previous studies of LynA have been hampered by knockout models that lack both LynA and LynB. To understand the role LynA plays in immune development and activation, we used CRISPR-Cas9 gene editing to generate novel mouse strains that express either LynA or LynB. Preliminary studies in these mice have demonstrated novel roles for LynA. Notably, LynA−/− mice have an enrichment in splenic dendritic cells and alveolar macrophages. Thus, LynA is not only an important regulator of myeloid cell activation, tuning cell-specific signaling thresholds, but also helps regulate the function of these cells. Further studies will investigate the impact of LynA deletion in myeloid inflammation sensing.

T-helper signals restore B-cell receptor signaling in autoreactive anergic B cells by upregulating CD45 phosphatase activity

We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells). We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus. Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca(2+) mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN, SYK, and CD45 mRNA. T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive Bcells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects. Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive Bcells in contributing to autoimmunity if not properly controlled.

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.

Increasing Expression Of The Efflux Transporter ABCB1 May Predispose CML Cells To Overt TKI Resistance

Tyrosine kinase inhibitors (TKIs) result in excellent responses in most Chronic Myeloid Leukemia (CML) patients. However, up to 35% of patients treated with imatinib (IM) exhibit resistance and more recently nilotinib (NIL) and dasatinib (DAS) resistance have also been observed. Mutations in the BCR-ABL kinase domain (KD) are the main cause of secondary TKI resistance. Other mechanisms include overexpression of BCR-ABL, LYN and ABCB1. Predicting patients with susceptibility to mutation development and disease progression is crucial, thus we investigated the kinetics of TKI resistance emergence in vitro and in vivo.ABCB1 is implicated in TKI efflux hence we postulated that overexpression of ABCB1 leads to reduced intracellular TKI concentrations, resulting in inferior inhibition of Bcr-Abl predisposing cells to resistance development. Accordingly, 3 CML blast crisis (BC) cell lines (K562, K562-Dox, KU812) were cultured in increasing concentrations of IM to 2 μM, NIL to 2 μM and DAS to 200 nM until we observed overt resistance defined as a significant increase in survival in cytotoxicity assays and p-Crkl dependent IC50. Mechanisms of resistance were investigated in cell line intermediates: BCR-ABL, ABCB1 and LYN mRNA expression levels were determined by RT-PCR and KD mutation sequencing was performed.In our TKI resistant cell lines (Table 1), an increase in ABCB1 mRNA was the initial change observed prior to the development of additional resistance mechanisms (KD mutations, ABCB1 BCR-ABL and LYN overexpression). Interestingly, in 4/6 cells lines ABCB1 mRNA reduced to basal levels or below following establishment of these additional resistance mechanisms.ABCB1 levels were assessed in 37 de novo CML patients treated with IM who achieved major molecular response (MMR) compared with patients who progressed to BC, lost MMR or developed KD mutations. ABCB1 levels were determined in blood at diagnosis and following therapy (selected patients summarized in Table 2). A sustained >2 fold rise in ABCB1 was observed prior to disease progression in 3/3 patients and in 13/16 patients who did not achieve MMR. Importantly, the same was not observed in patients who achieved MMR (1/6 patients). The fold change of ABCB1 mRNA at day 22 vs diagnosis in patients achieving MMR was significantly different to that in patients not achieving MMR (p=0.004). ABCB1 increased by >2 fold post therapy and decreased following mutation development in 3/12 patients, confirming observations made in vitro, while 6/12 patients demonstrated sustained increase in ABCB1 post mutation similar to results observed in progression patients. ABCB1 mRNA did not change during therapy in 3/12 patients with mutations. While we recognize the majority of cells present in patients who achieve MMR are normal rather than leukemic, it is important to note that in patients who do not achieve MMR, ABCB1 expression increases in the remaining leukemic cells.We conclude ABCB1 overexpression acts as an initial mediator of resistance, providing a favorable environment for development of further resistance. Sustained increased levels of ABCB1 may contribute to disease progression and lack of response to IM. Additionally, ABCB1 may serve as a prognostic indicator (eg: level at day 22) and potentially assist in development of treatment strategies using TKIs in combination with other medications to enhance intracellular TKI concentration. Disclosures:Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

Abstract 3563: Deep kinome sequencing identifies a novel D189Y mutation in the Src family kinase LYN as a possible mediator of antiestrogen resistance in ER+ breast cancer .

Abstract Estrogen receptor-positive (ER+) breast tumors adapt to hormone deprivation and acquire resistance to aromatase inhibitors (AIs). The proliferative rate of tumor cells measured by Ki67 after short-term treatment with an AI has been proposed as a surrogate endpoint of long term patient outcome. To identify kinase mutations associated with resistance to endocrine therapy, we performed deep-kinome sequencing on 4 ER+/HER2– breast tumors that retained high Ki67 scores (14.8 to 24.5%) after two weeks of pre-surgical therapy with letrozole. Genomic DNA from the surgically excised tumors was deep sequenced using a capture approach with oligonucleotides hybridizing to 612 genes, including 517 kinases with ≥300x coverage. All 4 tumors contained an activating mutation in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K. One tumor also contained a novel D189Y (565G>T) somatic mutation in the SH2 domain of LYN, a member of the Src family kinases (SFKs; variant frequency of 8%). Reverse-phase protein array (RPPA) analysis available in 10 tumors in this study revealed a significant correlation (p=0.006) between Y416 P-Src (which detects all SFKs) and a high post-letrozole Ki67 score. A siRNA screen targeting 779 kinases identified LYN as one of the top hits whose knockdown significantly reduced growth of ER+ MCF-7 cells with acquired resistance to estrogen deprivation. LYN mRNA expression was also upregulated in these cells. We next analyzed the Cancer Genome Atlas (TCGA) SNP data to detect copy number changes for 444 tumors where corresponding gene expression data were available. Copy number increases (>0.8 log2 ratio over normal matched DNA) in LYN were present in approximately 10% of breast cancers, with the highest copy number gains observed in luminal B tumors. In contrast, other SFKs showed less frequent copy number increases (YES1 <1%, SRC 2%, FYN 1%). Finally, we investigated the role of D189Y LYN in antiestrogen-resistant breast cancer using ER+/PIK3CA mutant MCF-7 cells transduced with GFP (control), wild-type (WT) LYN or D189Y LYN vectors. Overexpression of WT or mutant LYN resulted in increased phosphorylation of Src (at Y416), FAK, IGF-IR, EGFR, STAT3, AKT and MAPK. LYND189Y increased phosphorylation of FAK, EGFR, and IRS-1 as compared to LYNWT. Although stable transduction of either LYNWT or LYND189Y accelerated MCF-7 cell growth in estrogen-depleted medium, the mutant was more potent than LYNWT at inducing this effect. Further, LYND189Y overexpression rendered treatment with the ER downregulator fulvestrant or the PI3K inhibitor BKM120 less effective, as well as rescued apoptosis induced by BKM120. These results suggest, first, that LYN plays a role in escape from estrogen deprivation in a subset of ER+ breast cancers. Second, ER+ breast cancers harbor multiple molecular alterations capable of mediating hormone-independent growth. Citation Format: Emily M. Fox, Justin M. Balko, Carlos L. Arteaga. Deep kinome sequencing identifies a novel D189Y mutation in the Src family kinase LYN as a possible mediator of antiestrogen resistance in ER+ breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2013-3563

Expression levels of Lyn, Syk, PLCγ2 and ERK in patients with chronic lymphocytic leukemia, and higher levels of Lyn are associated with a shorter treatment-free survival

There is now strong evidence that B-cell receptor (BCR) signaling plays a major role in the development of chronic lymphocytic leukemia (CLL). The purpose of this study was to investigate the expression levels of some of the molecules involved in the signaling cascade originating from the BCR (Syk, Lyn, PLCγ2, ERK) and analyze possible correlations of mRNA levels with biological/clinical features. Our study population consisted of 92 consecutive patients with newly diagnosed CLL. Several genes of BCR signaling (Lyn, Syk, PLCγ2 and ERK) and ZAP-70 were measured by quantitative polymerase chain reaction (qPCR). The signaling molecules of BCR were strongly associated with each other, and ZAP-70 correlated well with Lyn, Syk, PLCγ2 and ERK. Associations between treatment response and Lyn, Syk, PLCγ2 and ERK were not found. Moreover, a higher level of Lyn mRNA was associated with a shorter treatment-free survival (TFS) (univariate analysis only; multivariate Cox analysis showed that only ZAP-70 and Binet stage were independent prognostic factors and associated with TFS). Though Lyn was not an independent prognostic factor, it still might be a new therapy target of CLL. BCR signaling provides perspectives for future development of an exciting new class of kinase inhibitors.

Promoter CpG methylation status in porcine Lyn is associated with its expression levels

Resistance to disease and improvement of product quality are important goals in pig farming. Tyrosine Protein Kinase Lyn (LYN) is one of several Src-family tyrosine kinases in immune cells. This protein functions both as a positive and negative regulator of B cell activation, and regulates signaling pathways through phosphorylation of inhibitory receptors, enzymes and adaptors, which suggested that LYN could be correlated with immunity and can be considered as a candidate gene to study in disease resistance. Until now, the profiles of expression and transcriptional regulation of the LYN gene in pig breeds different in immune capacity remain unclear. Using real-time PCR, it indicated that porcine LYN mRNA expressed mainly in immune organs including the spleen, duodenum and liver. Furthermore, Dahuabai pigs (a kind of Chinese indigenous pig breeds with higher immune capacity) showed significant higher LYN mRNA expression levels than that in Landrace. Methylation analysis indicates that LYN expression levels were associated with the methylation status of the LYN promoter, and methylation of the novel CpG site at −1268C/−1267G generated by transposition at −1267 (A→G) results in up-regulating transcriptional activity of this gene. Interestingly, the base A located in −1267 mainly exhibited in landrace while the base G mainly in Dahuabai pigs. These results might contribute to study the function of this gene in pig breeding for disease resistance.

Expression of lyn gene in Kaposi's sarcoma

Objective To analyse the expression of lyn gene in Kaposi's sarcoma(KS).Methods Immunohistochemical method(Envision)was used to detect the protein expression of lyn in tissue samples from 17 patients with KS and 17 normal human controls, and real-time PCR to measure the mRNA expression of lyn in fresh specimens from the tumor and adjacent normal tissues of 5 patients with KS.Results The lyn protein was cytoplasmically expressed in 17(100%)patients with KS and 2(11.8%)normal controls(x~2 =27.57, P ＜ 0.01).The expression intensity of lyn mRNA was significantly higher in fresh tumor tissues than in adjacent normal tissues(t = 2.82, P ＜ 0.05).Conclusion The abnormal expression of lyn may play an important role in the development of KS. Key words: Sarcoma,Kaposi; lyn; Gene expression

Opposite expression pattern of Src kinase Lyn in acute and chronic haematological malignancies

Lck/yes-related novel (Lyn) tyrosine kinase overexpression has been suggested to be important for leukaemic cell growth making it an attractive target for therapy. By contrast, Lyn deficiency was shown to be responsible for a phenotype resembling myeloproliferative neoplasm (MPN) in mice. We aimed to shed more light on Lyn's role in haematological neoplasm and systematically investigated Lyn expression in MPN, acute and chronic leukaemia subtypes (n = 236). On top, B-cell chronic lymphocytic leukaemia (B-CLL) and chronic myeloid leukaemia significantly overexpressed Lyn when compared to de novo acute lymphoblastic leukaemia, de novo acute myeloid leukaemia (AML) and Philadelphia-chromosome-negative myeloproliferative neoplasms (p < 0.001). Most of acute leukaemia subtypes showed a notable down-regulation of Lyn mRNA but anyhow individual cases were labelled for the active form of Lyn protein. Intriguingly, secondary AML evolved in myelodysplastic syndromes revealed almost undetectable Lyn. Overexpression of Lyn in B-CLL was associated with a significant down-regulation of microRNA-337-5p suggesting that aberrant expression of this particular microRNA could be involved in post-transcriptional control of Lyn mRNA fate. We conclude that tyrosine kinase Lyn contributes to the malignant phenotype in certain leukaemia subtypes and therefore attracts targeted therapy.

Expression patterns of Src-family tyrosine kinases during Xenopus laevis development

Src family tyrosine kinases (SFKs) play important roles in cell morphology, differentiation, motility and proliferation. Elevated expression and/or specific activity of Src kinases are characteristic for several types of human cancer. However, little information is available about the role and spatio-temporal expression of SFKs in early embryonic development. In this study we characterized, in Xenopus laevis, the expression patterns of five SFK genes src, fyn, yes, lyn and laloo as well as of the csk gene, a negative regulator of SFKs, using RT-qPCR and in situ hybridisation. We found that transcripts of all SFKs and csk were already detectable in one-cell embryos and their levels similarly oscillated during subsequent development. First, after stage 8, the levels of SFK and csk mRNAs began to decrease, reached a minimum between stages 10 and 28 and increased again. In the later stages (33-45), the levels of fyn, yes and csk mRNAs returned to approximately maternal ones, whereas the src, laloo and lyn mRNA transcripts exceeded, up to about 3.5-6-fold, their maternal levels. In situ hybridisation analysis located the SFK and csk transcripts in the animal hemisphere of Xenopus embryos. Subsequent gastrula stages showed signals in ectodermal cells, mid-neurula stage embryos at neural folds, and the tailbud stages showed strong signals in the brain and neural tube. RT-qPCR concentration profiling along the animal-vegetal axis proved in blastula and gastrula the preferential localisation of yes, src, lyn and csk transcripts towards the animal pole in a gradient-like manner. In contrast, laloo and fyn displayed a vegetal pole preference.

Evidence that Resistance to Nilotinib May Be Due to BCR-ABL, Pgp, or Src Kinase Overexpression

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl-positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance.

Lyn Kinase Overexpression Is One of the Mechanisms of Resistance to Nilotinib In Chronic Myeloid Leukemia

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Three nilotinib-resistant cell lines were obtained from the Ph-positive cell lines AR230, LAMA84 and K562. Over expression of the BCR-ABL gene was found in two nilotinib-resistant cell lines and the multidrug resistance gene (MDR-1) was found overexpressed in one of them, i.e, LAMA84 nilotinib resistant cell. The K562/DOX cell line, that displays resistance to several drugs by over expressing Pgp, was resistant to nilotinib, and this was reversed by simultaneous incubation with either verapamil or PSC833 confirming that nilotinib is a substrate of the Pgp. In one nilotinib-resistant cell line (K562-rn), we found over expression of p53/56 Lyn kinase, both at the mRNA and protein level (10- fold), and these cells were compared to their sensitive counterpart. Lyn silencing by siRNA restored sensitivity to nilotinib. Two Src kinase inhibitors (PP1 and PP2) partially restored sensitivity to nilotinib, but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, an inhibitor of Abl and Src-family kinases, inhibited phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line which over expressed p53/56 Lyn. Of 7 nilotinib-resistant CML patients, failure of nilotinib treatment was accompanied by mutations in Bcr-Abl kinase domain in 2 patients and an increase of Lyn mRNA expression (RQ-PCR) in 2 other patients. As an approach to confirm the involvement of the Lyn signalling pathway in nilotinib-resistance, we have used the Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technique. The tyrosine phosphorylated protein fraction was analyzed by tandem mass spectrometry. Peptide sequencing and quantification in the nilotinib-resistant cell line identified >350 proteins, of which several were hyper-phosphorylated; functional analysis of the different candidates is in progress.In conclusion, mechanisms of resistance to nilotinib in imatinib-resistant cell lines resemble those operating in CML patients, and up-regulated Lyn kinase signalling can play an important role in nilotinib resistance.

Establishment and characterization of a novel imatinib‐sensitive chronic myeloid leukemia cell line MYL, and an imatinib‐resistant subline MYL‐R showing overexpression of Lyn

In chronic myeloid leukemia (CML), resistance to imatinib is diverse. In addition to BCR-ABL-dependent mechanisms, BCR-ABL-independent mechanisms have been proposed. Here we established and characterized novel CML cell lines, an imatinib-sensitive cell line, MYL, and an imatinib-resistant subline, MYL-R. Treatment with imatinib inhibited phosphorylation of BCR-ABL and CrkL in both MYL and MYL-R, even though imatinib-induced apoptosis was preferentially observed in MYL than MYL-R, indicating that the resistance is based on a BCR-ABL-independent mechanism. MYL-R showed elevated expressions of Lyn mRNA, Lyn protein, phosphorylated Lyn, and phosphorylated STAT5. Silencing of Lyn by short-interfering RNA (siRNA) in MYL-R, but not in MYL, induced significant growth-inhibition, increased caspase-3 activity, and induced partial recovery from imatinib-resistance. Expression of Bcl-2, previously reported to be associated with Lyn-mediated resistance, was not elevated in MYL-R. Expression of Bim, which plays an important role in imatinib-induced cell-killing, was not suppressed in MYL-R. These results imply that diverse mechanisms of resistance exist among cell types. Treatment of MYL-R cells with various reagents known to have anti-leukemic activity revealed that zoledronic acid and the farnesyl transferase inhibitor (SCH 66336) showed strong synergism with imatinib; interferon alpha, PP2, CGP76030, and FK228 (depsipeptide) showed synergism; whereas soluble TRAIL and As2O3 showed additivity or antagonism, and 17-AAG and radicicol showed antagonism. Treatment with either PP2 or zoledronic acid induced greater growth-reduction in MYL-R than MYL. Taken together, Lyn may play an important role in imatinib-resistance in MYL-R. Some novel reagents, including siRNA targeting Lyn, may have good potential to overcome this resistance.

Use of Imatinib Mesylate for Favorable Control of Hypercalcemia Mediated by Parathyroid Hormone-Related Protein in a Patient with Chronic Myelogenous Leukemia at Blast Phase

The case of a 72-year-old woman with chronic myelogenous leukemia in blast phase (BP) with hypercalcemia is reported. Bone x-ray examination revealed multiple osteolytic lesions throughout the body. The serum level of parathyroid hormone-related protein (PTHrP) was elevated, and PTHrP messenger RNA (mRNA) was detectable in the peripheral blood mononuclear cells (PBMNC) at BP but was not detectable at chronic phase (CP).Treatment with conventional chemotherapy did not completely control either serum calcium level or serum PTHrP level. Treatment with imatinib mesylate (imatinib) alone rapidly normalized these parameters in parallel with a decrease in the number of blast cells. The treatment also maintained the patient in good condition for approximately 3 months, even though the number of blast cells, serum calcium level, serum PTHrP level, and PTHrP mRNA level increased at the terminal stage. Mutations of the p53, K-Ras, and BCR-ABL genes in PBMNC at BP were absent. A noteworthy feature in this patient was that PBMNC at BP but not at CP showed high Lyn mRNA expression. Taken together the findings showed that production of PTHrP by blast cells was favorably controlled by imatinib therapy alone. Imatinib may prolong survival time at BP even though the patients have the complication of PTHrP-mediated hypercalcemia.

B-Cell Kinase Lyn Deficiency in Patients With Systemic Lupus Erythematosus

BackgroundTo better understand the molecular background of B-cell overactivity characterizing systemic lupus erythematosus (SLE), we examined the expression of the CD22 co-receptor and of kinase Lyn, which are involved in...

Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells.

Erythropoietin stimulates the immature erythroid J2E cell line to terminally differentiate and maintains the viability of the cells in the absence of serum. In contrast, a mutant J2E clone (J2E-NR) fails to mature in response to erythropoietin; however, it remains viable in the presence of the hormone. We have shown previously that intracellular signalling is disrupted in the J2E-NR cell line and that tyrosine phosphorylation is dramatically reduced after erythropoietin stimulation. In this study we investigated the defect in J2E-NR cells that is responsible for their inability to differentiate. Screening of numerous signalling molecules revealed that the lyn tyrosine kinase appeared to be absent from J2E-NR cells. On closer examination, both lyn mRNA and protein content were reduced >500-fold. Consistent with a defect in lyn, amphotropic retroviral infection of J2E-NR cells with lyn restored the ability of the cells to synthesize haemoglobin and enabled the cells to mature morphologically. Conversely, the ability of J2E cells to differentiate in response to epo was severely curtailed when antisense lyn oligonucleotides or a dominant negative lyn were introduced into the cells. However, erythropoietin-supported viability was unaffected by reducing lyn activity. The ability of two other erythropoietin-responsive cell lines (R11 and R24) to differentiate in response to the hormone was also reduced by dominant negative lyn. Finally, co-immunoprecipitation and yeast two-hybrid analyses indicated that lyn directly associated with the erythropoietin receptor complex. These data indicate for the first time an essential role for lyn in erythropoietin-initiated differentiation of J2E cells but not in the maintenance of cell viability.

P 53 and p56: A new signaling pathway in human endometrium and endometrial adenocarcinomas

To identify specific tyrosine kinases that are involved in endometrial signaling and to study their in vivo expression in normal and abnormal endometrium. We hypothesized that proteins that are differentially expressed would be more likely to be important in regulated cellular events. Complementary DNA libraries, constructed from human secretory (n = 5) and proliferative (n = 5) endometrial specimens, were screened with a polyclonal anti-phosphotyrosine antibody. Positive clones were sequenced and screened for differential expression using immunoblotting and Northern analysis of samples from proliferative and secretory endometrium. The expression of one identified clone, lyn, a Src family member, was characterized further with Western and Northern blot analyses and immunolocalization. One protein identified by the above method was lyn, a member of the src family of protein tyrosine kinases, never before described in the human endometrium. Western blot analysis revealed two forms of lyn protein, p53lyn and p56lyn, that were most abundant in the late secretory phase. Immunohistochemistry demonstrated uniform protein expression by all cells in normal glandular epithelium and suggested a correlation between lyn protein expression and cell differentiation for human endometrial adenocarcinomas, with markedly-elevated levels noted in poorly differentiated adenocarcinomas compared with well-differentiated tumors (n = 3). Northern hybridization confirmed the presence of the expected 3.5-kb lyn transcript in normal and abnormal endometrium. Our data demonstrate that human cDNA libraries created from different phases of the menstrual cycle can be screened successfully using anti-phosphotyrosine antibodies to identify differentially expressed protein tyrosine kinases. Although p53lyn and p56lyn expression has been thought of as a predominantly lymphoid-specific tyrosine kinase, we show prominent expression of lyn protein and mRNA by normal and malignant epithelium of the human endometrium, suggesting a role in endometrial signaling and human reproduction.

The non-receptor tyrosine kinase Lyn is localised in the developing murine blood-brain barrier

The blood-brain barrier, formed by brain endothelium, is critical for brain function. The development of the blood-brain barrier involves brain angiogenesis and endothelial cell differentiation, processes which require active signal transduction pathways. The differentiation of brain endothelial cells to the “blood-brain-barrier phenotype” involves cytoskeletal changes which modulate the tightness of the barrier. In order to identify signal transduction proteins involved in blood-brain barrier development, cDNA from bovine and murine brain endothelial cells was used in a polymerase chain reaction for cloning of DNA encoding Src homology 3 domains. Src homology 3 domains are structural domains found in many signal transduction proteins. These domains often mediate interaction of signaling proteins with the cytoskeleton and therefore may play a role in the regulation of the cytoskeletal changes which occur during blood-brain-barrier development. Unexpectedly, all bovine and murine clones analyzed from polymerase chain reactions encoded the Src homology 3 domain of one protein, namely the non-receptor tyrosine kinase, Lyn, which is involved in signal transduction in cells of the hemopoietic system. In situ hybridization analyses confirmed the presence of lyn mRNA in developing blood vessels in embryonic and early post-natal mouse brain, but not in endothelium outside the brain. In bovine brain endothelial cells in primary culture, p53 lyn is highly abundant and present in two forms which have different patterns of tyrosine phosphorylation. These data suggest that Lyn may be involved in transduction of growth and differentiation signals required for blood-brain-barrier development.