Lyn Expression Research Articles

MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis.

Gastric cancer (GC) is the fourth leading cause of cancer-related death worldwide. Minichromsome maintenance proteins family member 8 (MCM8) assists DNA repair and DNA replication. MCM8 exerts tumor promotor function in multiple digestive system tumors. MCM8 is also considered as a potential cancer therapeutic target. Bioinformatics methods were used to analyze MCM8 expression and clinicopathological significance. MCM8 expression was detected by immunohistochemistry (IHC) staining and qRT-PCR. MCM8 functions in GC cell were explored by Celigo cell counting, colony formation, wound-healing, transwell, and annexin V-APC staining assays. The target of MCM8 was determined by human gene expression profile microarray. Human phospho-kinase array kit evaluated changes in key proteins after ribosomal protein S15A (RPS15A) knockdown. MCM8 functions were reassessed in xenograft mouse model. IHC detected related proteins expression in mouse tumor sections. MCM8 was significantly upregulated and predicted poor prognosis in GC. High expression of MCM8 was positively correlated with lymph node positive (p < 0.001), grade (p < 0.05), AJCC Stage (p < 0.001), pathologic T (p < 0.01), and pathologic N (p < 0.001). MCM8 knockdown inhibited proliferation, migration, and invasion while promoting apoptosis. RPS15A expression decreased significantly after MCM8 knockdown. It was also the only candidate target, which ranked among the top 10 downregulated differentially expressed genes (DEGs) in sh-MCM8 group. RPS15A was identified as the target of MCM8 in GC. MCM8/RPS15A promoted phosphorylation of P38α, LYN, and p70S6K. Moreover, MCM8 knockdown inhibited tumor growth, RPS15A expression, and phosphorylation of P38α, LYN, and p70S6K invivo. MCM8 is an oncogene and predicts poor prognosis in GC. MCM8/RPS15A facilitates GC progression.

A novel lncRNA associated with the prognosis of patients with colorectal cancer resists apoptosis through the LYN/BCL-2 pathway

PurposeWe found a novel lncRNA named lncAC138150.2 related to the overall survival and staging of patients with colorectal cancer (CRC) by bioinformatic analysis using data from the Cancer Genome Atlas (TCGA), and the study aimed to elucidate the function of lncAC138150.2 and underlying mechanisms. MethodsTarget molecules were knocked down by transfection with antisense oligonucleotides (ASOs), siRNAs, or lentiviruses and overexpressed by transfection with plasmids. The function of lncAC138150.2 was determined using histological, cytological, and molecular biology methods. The underlying mechanism of lncAC138150.2 function was investigated using RNA-seq, bioinformatics analysis, and molecular biology methods. ResultsThe expression of lncAC138150.2 was increased in colorectal tissues compared with paired normal tissues. The lncAC138150.2 knockdown increased apoptosis but did not change the cell proliferation, cell cycle distribution, or cell migration ability of CRC cells, while lncAC138150.2 overexpression decreased CRC apoptosis. lncAC138150.2 was mainly located in the cell nucleus, and each lncAC138150.2 transcript knockdown increased CRC apoptosis. BCL-2 pathway was significantly altered in apoptosis induced by lncAC138150.2 knockdown, which was alleviated by BAX knockdown. The expression of LYN was significantly decreased with lncAC138150.2 knockdown, LYN knockdown increased CRC apoptosis, and its overexpression completely alleviated CRC apoptosis induced by lncAC138150.2 knockdown. ConclusionlncAC138150.2 significantly inhibited CRC apoptosis and affected the prognosis of patients with CRC, through the LYN/BCL-2 pathway.

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.

Expression of USP25 associates with fibrosis, inflammation and metabolism changes in IgG4-related disease

IgG4-related disease (IgG4-RD) has complex clinical manifestations ranging from fibrosis and inflammation to deregulated metabolism. The molecular mechanisms underpinning these phenotypes are unclear. In this study, by using IgG4-RD patient peripheral blood mononuclear cells (PBMCs), IgG4-RD cell lines and Usp25 knockout mice, we show that ubiquitin-specific protease 25 (USP25) engages in multiple pathways to regulate fibrotic and inflammatory pathways that are characteristic to IgG4-RD. Reduced USP25 expression in IgG4-RD leads to increased SMAD3 activation, which contributes to fibrosis and induces inflammation through the IL-1β inflammatory axis. Mechanistically, USP25 prevents ubiquitination of RAC1, thus, downregulation of USP25 leads to ubiquitination and degradation of RAC1. Decreased RAC1 levels result in reduced aldolase A release from the actin cytoskeleton, which then lowers glycolysis. The expression of LYN, a component of the B cell receptor signalosome is also reduced in USP25-deficient B cells, which might result in B cell activation deficiency. Altogether, our results indicate a potential anti-inflammatory and anti-fibrotic role for USP25 and make USP25 a promising diagnostic marker and potential therapeutic target in IgG4-RD.

476 Expression and distribution of Src kinase LYN in placenta of patients with preeclampsia (PE)

476 Expression and distribution of Src kinase LYN in placenta of patients with preeclampsia (PE)

Conditional in vivo deletion of LYN kinase has little effect on a BRCA1 loss-of-function-associated mammary tumour model.

LYN kinase is expressed in BRCA1 loss-of-function-dependent mouse mammary tumours, in the cells of origin of such tumours, and in human breast cancer. Suppressing LYN kinase activity in BRCA1-defective cell lines as well as in in vitro cultures of Brca1-null mouse mammary tumours is deleterious to their growth. Here, we examined the interaction between LYN kinase and BRCA1 loss-of-function in an in vivo mouse mammary tumour model, using conditional knockout Brca1 and Lyn alleles. Comparison of Brca1 tumour cohorts showed little difference in mammary tumour formation between animals that were wild type, heterozygous or homozygous for the conditional Lyn allele, although this was confounded by factors including incomplete Lyn recombination in some tumours. RNA-sequencing analysis demonstrated that tumours with high levels of Lyn gene expression had a slower doubling time, but this was not correlated with levels of LYN staining in tumour cells themselves. Rather, high Lyn expression and slower tumour growth were likely a result of B-cell infiltration. The multifaceted role of LYN indicates that it is likely to present difficulties as a therapeutic target in breast cancer.

Abstract PR02: Lyn kinase regulates myeloid-cell responses to mammary tumors

Abstract Myeloid-cell enrichment in tumors is correlated with increased metastasis, reduced survival times, and poor treatment outcomes in multiple cancers, including breast cancer. Many signals (CSF, Il-6, chemokine) to myeloid cells in the tumor microenvironment are transduced through the Src-family kinase Lyn. Expression of both isoforms of Lyn (LynA and LynB) is regulated by cytokine signaling and cell type, and total Lyn expression is negatively correlated with breast cancer patient survival. Lyn expression in macrophages contributes to leukemogenesis, but the functional contributions of LynA and LynB in solid tumors have not been studied. We are examining the contributions of Lyn in trafficking of myeloid cells to mammary tumors, pro-tumor polarization of tumor-associated macrophages, and growth and metastasis of mammary tumors. In an E0771 orthotopic transplant model in hosts expressing or lacking LynA or LynB expression, we have discovered that Lyn expression in the mammary tumor microenvironment promotes the early recruitment of monocytes, macrophages, neutrophils, and dendritic cells, as determined by flow cytometry analysis of tumors early in development. Furthermore, Lyn promotes pro-tumor polarization of tumor-associated macrophages. Tumors from Lyn knockout animals contained populations of inflammatory, anti-tumor macrophages that were not significantly different in size from WT, but the CD206+, anti-inflammatory population was reduced. Finally, we have observed that expression of either Lyn isoform in the tumor microenvironment can promote tumor growth and decrease survival in orthotopically transplanted mice. In contrast, our studies suggest that only LynB promotes metastasis to the lungs of transplanted mice, as determined by in vivo imaging of a hyper-metastatic, luciferase-expressing variant of E0771. Together, our findings suggest that Lyn kinase plays a role in the signaling response of myeloid cells, which are major factors in disease course and treatment response, to tumor-associated stimuli. Modulation of Lyn activity, specifically the LynB isoform, or the activity of its downstream signaling partners, is thus a potential strategy for influencing the trafficking and polarization of myeloid cells within breast tumors to benefit patients. Citation Format: Joseph T Greene, Ben F Brian, Aditi Bapat, Tanya S Freedman. Lyn kinase regulates myeloid-cell responses to mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR02.

Upregulation of the protein kinase Lyn is associated with renal injury in type 2 diabetes patients

Background The role of inflammation in the pathogenesis of type 2 diabetes mellitus (T2DM) is well established. Lyn, a member of the nonreceptor protein tyrosine kinase Src family, has been reported to modulate inflammatory signaling pathways. Methods Lyn expression was assessed in kidney biopsies of 11 patients with diabetic kidney disease (DKD) and in kidney tissues of streptozotocin (STZ)-induced DKD mice. 102 recruited T2DM patients were divided into three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. Twenty-one healthy volunteers were recruited as a control group. Clinical data, blood and urine samples of all individuals were collected for analysis. Results Lyn expression was augmented in the kidneys of DKD patients and STZ-induced diabetic mice. Compared with control and normoalbuminuria groups, both mRNA and protein expression of Lyn in peripheral blood mononuclear cells (PBMCs) in the macroalbuminuria group were significantly increased (p < .05). Elevated Lyn levels were independently related to urine albumin/urine creatinine ratio and were positively associated with key inflammatory factors, namely interleukin-1β, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Additionally, Lyn exhibited a noteworthy connection with renal tubular injury indicators, specifically urinary neutrophil gelatinase-associated lipocalin and urinary retinol binding protein. ROC curve analysis showed that Lyn could predict albuminuria in diabetic patients with an area under the curve of 0.844 (95% CI: 0.764–0.924). Conclusion Lyn levels in PBMCs exhibited a positive correlation with the severity of albuminuria, renal tubular damage, and inflammatory responses. Hence, Lyn may be a compelling candidate for predicting albuminuria levels in diabetes.

Integrated proteomics and phosphoproteomics analyses of esophageal cancer cells with different invasive abilities

AimsEsophageal squamous cell carcinoma (ESCC) is one of the aggressive and lethal malignancies with an extremely poor prognosis. It is necessary to explore the molecular mechanisms of ESCC invasion. Main methodsWe utilized high-throughput mass spectrometry to analyze the proteomes and phosphorylation profiles of two ESCC cell lines with differing invasion capacities (HK vs TE10). Differentially expressed proteins and phosphorites were identified, followed by comprehensive bioinformatics analyses encompassing function and pathway enrichment, protein-protein interaction (PPI) network analysis, hub gene identification, co-expression analysis, kinase-substrate prediction, and drug-target network analysis. CCK-8 assay, transwell examination, wound-healing assay, and western blot was used to validate the effects of fostamatinib on ESCC cells proliferation, invasion, migration, and LYN expression. Key findingsThe Q4 cluster of differentially phosphorylated proteins was primarily associated with functions and pathways relevant to tumor metastasis. Phosphorylated hub proteins including ARHGAP35, CTNNA1, and SHC1 were identified through the analysis of PPI network, and their respective regulated kinases were predicted. Among the predicted kinases, LYN was validated to be associated with lymph node metastasis (N0 vs. N1–3) and prognosis in ESCC patients at mRNA levels using TGGA data and protein levels in ESCC tissues (p < 0.05). Validation experiments confirmed the inhibitory effects of fostamatinib on ESCC cells proliferation, migration, invasion, and LYN expression. SignificanceOur multi-omics analysis offers deeper perspectives on ESCC invasiveness and unveils new phosphorylated hub proteins with their regulatory kinase. This study also suggests that fostamatinib may be a potential agent for treating ESCC.

Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.

The pro-inflammatory phenotype of microglia usually induces neuroinflammatory reactions in neuropathic pain. Glycometabolism shift to glycolysis can promote the pro-inflammatory phenotype transition of microglia. The omics data analysis suggest a critical role for Lyn dysregulation in neuropathic pain. The present study aimed at exploring the mechanism of Lyn-mediated glycolysis enhancement of microglia in neuropathic pain. Neuropathic pain model was established by chronic constriction injury (CCI), then pain thresholds and Lyn expression were measured. Lyn inhibitor Bafetinib and siRNA-lyn knockdown were administrated intrathecally to evaluate the effects of Lyn on pain thresholds, glycolysis and interferon regulatory factor 5 (IRF5) nuclear translocation of microglia in vivo and in vitro. ChIP was carried out to observe the binding of transcription factors SP1, PU.1 to glycolytic gene promoters by IRF5 knockdown. Finally, the relationship between glycolysis and pro-inflammatory phenotype transition of microglia was evaluated. CCI led to the upregulation of Lyn expression and glycolysis enhancement in microglia of spinal dorsal horn. Bafetinib or siRNA-lyn knockdown intrathecally alleviated pain hyperalgesia, suppressed glycolysis enhancement and inhibited nuclear translocation of IRF5 in CCI mice. Also, IRF5 promoted the binding of transcription factors SP1, PU.1 to glycolytic gene promoters, and then the enhanced glycolysis facilitated the proliferation and pro-inflammatory phenotype transition of microglia and contributed to neuropathic pain. Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.

Enhanced Lyn Activity Causes Severe, Progressive Emphysema and Lung Cancer.

The epidemiological patterns of incident chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma are changing, with an increasing fraction of disease occurring in patients who are never-smokers or were not exposed to traditional risk factors. However, causative mechanism(s) are obscure. Overactivity of Src family kinases (SFKs) and myeloid cell-dependent inflammatory lung epithelial and endothelial damage are independent candidate mechanisms, but their pathogenic convergence has not been demonstrated. Here we present a novel preclinical model in which an activating mutation in Lyn, a nonreceptor SFK that is expressed in immune cells, epithelium, and endothelium-all strongly implicated in the pathogenesis of COPD-causes spontaneous inflammation, early-onset progressive emphysema, and lung adenocarcinoma. Surprisingly, even though activated macrophages, elastolytic enzymes, and proinflammatory cytokines were prominent, bone marrow chimeras formally demonstrated that myeloid cells were not disease initiators. Rather, lung disease arose from aberrant epithelial cell proliferation and differentiation, microvascular lesions within an activated endothelial microcirculation, and amplified EGFR (epidermal growth factor receptor) expression. In human bioinformatics analyses, LYN expression was increased in patients with COPD and was correlated with increased EGFR expression, a known lung oncogenic pathway, and LYN was linked to COPD. Our study shows that a singular molecular defect causes a spontaneous COPD-like immunopathology and lung adenocarcinoma. Furthermore, we identify Lyn and, by implication, its associated signaling pathways as new therapeutic targets for COPD and cancer. Moreover, our work may inform the development of molecular risk screening and intervention methods for disease susceptibility, progression, and prevention of these increasingly prevalent conditions.

Identification of biomarkers of hepatocellular carcinoma gene prognosis based on the immune-related lncRNA signature of transcriptome data

Long non-coding RNAs (lncRNAs) are well established to have an important role in cancer. The goal of this research was to investigate the prognostic usefulness of putative immune-related lncRNAs in hepatocellular carcinoma (HCC). The developed lncRNA signature was validated using 343 HCC patients from The Cancer Genome Atlas (TCGA) and 81 samples from Gene Expression Omnibus (GEO). Cox regression and Least Absolute Shrinkage and Selection Operator (LASSO) analysis were used to analyze immune-related lncRNAs for HCC prognosis. Patients in the low-risk group survived substantially longer than those in the high-risk group (P < 0.05). The discovered signal might be a useful prognostic factor for predicting patient survival. Overall survival predicted some clinical net improvements, according to the nomogram. Numerous enrichment approaches (including gene set enrichment analysis) were utilized to investigate the underlying mechanisms. Drug metabolism, mTOR, and p53 signaling pathways were associated with high-risk groups. When the expression of lncRNA PRRT3-AS1 was silenced in HepG2 cells, the proliferation, migration, and invasion abilities of HepG2 cells were decreased, and apoptosis was enhanced. In the supernatant from HepG2 cells with PRRT3-AS1 knockdown, the anti-inflammatory factors IL-10 and TGF-1 were induced, whereas the pro-inflammatory factors IL-1β, TNF-α, and IL-6 were reduced (P < 0.05). After PRRT3-AS1 knockdown, the protein expression of CD24, THY1, LYN, CD47, and TRAF2 in HepG2 cells was attenuated (P < 0.05). The discovery of five immune-related lncRNA signatures has significant therapeutic significance for predicting patient prognosis and directing personalized treatment for patients with HCC, which requires additional prospective confirmation.

Immune cell infiltration-related clinical diagnostic model for Ankylosing Spondylitis.

Background: The pathogenesis and diagnosis of Ankylosing Spondylitis (AS) has remained uncertain due to several reasons, including the lack of studies on the local and systemic immune response in AS. To construct a clinical diagnostic model, this study identified the micro RNA-messenger RNA (miRNA-mRNA) interaction network and immune cell infiltration-related hub genes associated with AS. Materials and Methods: Total RNA was extracted and purified from the interspinous ligament tissue samples of three patients with AS and three patients without AS; miRNA and mRNA microarrays were constructed using the extracted RNA. Bioinformatic tools were used to construct an miRNA-mRNA network, identify hub genes, and analyze immune infiltration associated with AS. Next, we collected the blood samples and clinical characteristics of 359 patients (197 with AS and 162 without AS). On the basis of the clinical characteristics and results of the routine blood tests, we selected immune-related cells whose numbers were significantly different in patients with AS and patients without AS. Univariate and multivariate logistic regression analysis was performed to construct a nomogram. Immunohistochemistry staining analysis was utilized to verify the differentially expression of LYN in AS and controls. Results: A total of 225 differentially expressed miRNAs (DE miRNAs) and 406 differentially expressed mRNAs (DE mRNAs) were identified from the microarray. We selected 15 DE miRNAs and 38 DE mRNAs to construct a miRNA-mRNA network. The expression of LYN, an immune-related gene, correlated with the counts of monocytes, neutrophils, and dendritic cells. Based on the independent predictive factors of sex, age, and counts of monocytes, neutrophils, and white blood cells, a nomogram was established. Receiver operating characteristic (ROC) analysis was performed to evaluate the nomogram, with a C-index of 0.835 and AUC of 0.855. Conclusion: LYN, an immune-related hub gene, correlated with immune cell infiltration in patients with AS. In addition, the counts of monocytes and neutrophils were the independent diagnostic factors for AS. If verified in future studies, a diagnostic model based on these findings may be used to predict AS effectively.

A dominant function of LynB kinase in preventing autoimmunity.

Here, we report that the LynB splice variant of the Src-family kinase Lyn exerts a dominant immunosuppressive function in vivo, whereas the LynA isoform is uniquely required to restrain autoimmunity in female mice. We used CRISPR-Cas9 gene editing to constrain lyn splicing and expression, generating single-isoform LynA knockout (LynAKO) or LynBKO mice. Autoimmune disease in total LynKO mice is characterized by production of antinuclear antibodies, glomerulonephritis, impaired B cell development, and overabundance of activated B cells and proinflammatory myeloid cells. Expression of LynA or LynB alone uncoupled the developmental phenotype from the autoimmune disease: B cell transitional populations were restored, but myeloid cells and differentiated B cells were dysregulated. These changes were isoform-specific, sexually dimorphic, and distinct from the complete LynKO. Despite the apparent differences in disease etiology and penetrance, loss of either LynA or LynB had the potential to induce severe autoimmune disease with parallels to human systemic lupus erythematosus (SLE).

Evaluation of the therapeutic potential of masitinib and expression of its specific targets c-Kit, PDGFR-α, PDGFR-β, and Lyn in canine prostate cancer cell lines.

Canine prostate cancer is classified into adenocarcinoma, transitional cell carcinoma with prostatic involvement, and mixed forms. Early metastatic spread leads to poor prognosis and limited treatment options. Masitinib is approved for the treatment of canine mast cell tumours and inhibits tyrosine kinase c-Kit, tyrosine-protein kinase Lyn (Lyn), and platelet-derived growth factor receptors alpha and beta (PDGFR-α, PDGFR-β), which are known to be expressed in canine prostate cancer. The aim of this study was to evaluate masitinib in an in vitro model consisting of cell lines from primary prostate adenocarcinoma, the associated lymph node metastasis of the same patient, and transitional cell carcinoma. To assess the suitability of the model system, the targets of masitinib were investigated by immunocytochemistry in the cell lines and by immunohistochemistry in the respective formalin-fixed, paraffin-embedded (FFPE) original neoplastic tissue. After exposure to masitinib, cell viability, cell count, apoptosis induction, and protein expression of c-Kit, Lyn, PDGFR-α, and PDGFR-β were assessed. To hedge the efficacy, two application protocols of masitinib (single application or 12-h double-dose regimen) were compared. Immunocytochemical and immunohistochemical analysis revealed increased Lyn, PDGFR-α, and PDGFR-β expression in cell lines and FFPE original neoplastic tissue compared to healthy prostate tissue. Masitinib exposure increased apoptosis, while the cell counts and cell viability decreased in a dose- and application interval-dependent manner, with increased impact in the 12-h double-dose regimen. These in vitro effects of masitinib in canine prostate cancer and associated metastasis support further in vivo research and modifications of the clinical treatment protocol in future studies.

Bioinformatics Analysis to Screen the Key Genes and Pathways in the Formation of Early T-cell Precursor Acute Lymphoblastic Leukemia

<sec><title>Purpose</title> Early T-cell precursor acute lymphoblastic leukemia (EPT-ALL) is a rare type of ALL that shows genetic characteristics of both ALL and acute myeloid leukemia (AML) but has a poorer prognosis and a higher recurrence rate. The aims of this study were to explore the underlying molecular mechanisms and specific biomarkers of EPT-ALL by bioinformatics analysis. </sec><sec><title>Methods</title> Two expression profile datasets (GSE8879 and GSE28703) were integrated to identify candidate genes of EPT-ALL. Differentially expressed genes (DEGs) between EPT-ALL and classic T-ALL were identified using edge R package. Protein–protein interaction (PPI) network clustering modules were analyzed with STRING and Cytoscape. In addition, the plugins of Cytoscape was used for hub gene functional enrichment analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and PPI network analysis. Survival analysis was performed by GEPIA and CCLE database. </sec><sec><title>Results</title> The GSE profiles shared 132 DEGs, including 63 upregulated genes and 69 downregulated genes. KEGG enrichment analysis of DEGs showed that the top 3 pathways were hematopoietic cell lineage, NF-kappaB signaling pathway and T cell receptor signaling pathway. Twenty-seven hub genes were identified from PPI by Cytoscape. High expression of ITGAM (integrin subunit alpha M) and LYN (LYN proto-oncogene) were associated with statistical significantly poorer overall survival rates by survival analysis using GEPIA database. Besides, LYN was differently expressed in EPT-ALL compared with classic T-ALL by searching in CCLE database. </sec><sec><title>Conclusion</title> We screened two hub genes which may consider as candidate novel biomarker in EPT. In summary, we elucidated LYN as a biomarker for the differentiation of EPT-ALL from classic T-ALL. </sec>

Evidence of off-target effects of bosutinib that promote retinoic acid-induced differentiation of non-APL AML cells

ABSTRACT In the present study, we determined the effects of the Src family kinase (SFK) inhibitor, Bosutinib, and the engineered loss of the Lyn SFK on all-trans retinoic acid-induced leukemic cell differentiation. Retinoic acid (RA) is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing differentiation of a non-APL AML cell model, the HL-60 human myeloblastic (FAB-M2) leukemia cell line, via activation of a novel signalsome containing an ensemble of signaling molecules that drive differentiation. Bosutinib is an inhibitor of SFKs used to treat myeloid leukemias where prominent high expression of SFKs, in particular Lyn, has been observed. Using either Bosutinib or loss of Lyn expression due to shRNA promoted RA-induced phenotypic differentiation, G0 arrest, and respiratory burst (functional differentiation) of HL-60 cells. Signaling events putatively seminal to RA-induced differentiation, the expression of Fgr, Cbl, Slp-76 and Vav, and the phosphorylation of c-Raf (pS259), Vav (p-tyr), and Slp76 (p-tyr) were not inhibited by Bosutinib or loss of Lyn. Nor was RA-induced upregulation of p-tyr phosphorylation of p47phox, a member of the NADPH complex that produces ROS, a putative phosphorylation dependent signaling regulator. Surprisingly, Bosutinib still works in the absence of Lyn to enhance RA-induced differentiation and neither compromised RA-induced expression, nor phosphorylation of signaling molecules that drive differentiation. These findings suggested there is a novel, off-target, Lyn-independent effect of Bosutinib that is of therapeutic significance to differentiation therapy.

Effects of G-Rh2 on mast cell-mediated anaphylaxis via AKT-Nrf2/NF-κB and MAPK-Nrf2/NF-κB pathways

BackgroundThe effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. MethodsAsthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. ResultsG-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1β and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1β, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. ConclusionG-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-κB and p38MAPK-Nrf2/NF-κB signaling pathways.

Berberine exerts a protective effect on rats with polycystic ovary syndrome by inhibiting the inflammatory response and cell apoptosis

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disease of the female reproductive system that seriously affects women’s health. Berberine (BBR) has many pharmacological properties and is used as an insulin sensitizer. This study aimed to investigate the effect of BBR on PCOS and explore its related mechanisms.MethodsForty-two rats were randomly divided into the following six groups (n = 7 per group): control, control + BBR, PCOS-normal diet (ND), PCOS-ND + BBR, PCOS-high-fat diet (HFD), and PCOS-HFD + BBR. The PCOS rat models were established by injecting rats with dehydroepiandrosterone. Further, the rats were gavaged with BBR (150 mg/kg/d) for 6 weeks. Then, the body weight, HOMA-IR, and testosterone levels of all rats were determined. Cell apoptosis of ovary granulosa cells was determined by a TUNEL assay kit. Real-time quantification PCR (RT-qPCR) and western blotting were utilized to evaluate the expression of TLR4, LYN, PI3K, Akt, NF-kB, TNF-α, IL-1, IL-6, and caspase-3.ResultsBBR reduced the levels of insulin resistance and testosterone in PCOS rats. Additionally, the cell apoptosis rate increased significantly in PCOS rats (P < 0.05) and decreased after BBR treatment (P < 0.05). The results of RT-qPCR and western blotting showed that the expression levels of TLR4, LYN, PI3K, Akt, NF-kB, TNF-α, IL-1, IL-6, and caspase-3 significantly increased in PCOS rats, while BBR suppressed their expression levels.ConclusionsBBR may relieve PCOS pathology and IR values by inhibiting cell apoptosis and by regulating the expression levels of TLR4, LYN, PI3K, Akt, NF-kB, TNF-α, IL-1, IL-6, and caspase-3.

Conditional CRISPR-Mediated Deletion of Lyn Kinase Promotes Megakaryocyte Differentiation, Platelet Production and Function

Thrombocytopenia leading to life-threatening excessive bleeding can be caused by defects in megakaryocyte/platelet production, platelet depletion by platelet-specific auto- or allo-antibodies, blood loss following trauma or surgery, or as a result of damage to bone marrow blood stem cells induced by chemo- or radiation therapy. Currently, platelets available for transfusion are entirely dependent on volunteer blood donation, which are often in short supply, carry a risk of infection, and often unable to be antigen-matched for alloimmunized patients. Thus, maintaining an adequate supply of platelets for transfusion is often a challenge. In response to this growing clinical need, a number of laboratories have started differentiating human induced pluripotent stem cells (iPSCs) into megakaryocytes, with the long-term goal of creating a readily available supply of antigen-matched, highly functional platelets. Current methods for generating platelets from iPS cell-derived megakaryocytes have been improved to produce enough platelets in vitro to begin phase I safety trials in humans; however, modifying the iPS cell genome has the potential to further improve both the yield and function of in vitro-generated platelets. The purpose of this investigation, therefore, was to combine recent advances in gene editing technology with iPS cell differentiation techniques to improve the yield and/or functionality of in vitro-derived platelet products. As a start, we sought to examine whether disruption of negative regulators of platelet production and/or activation might allow the generation of increased numbers of platelets with improved hemostatic effectiveness. One such negative regulator of platelet function is the Src family kinase, Lyn. Previous studies using pharmacologic inhibitors and global Lyn knockout mice have shown that that disrupting Lyn activity leads to mild thrombocytosis and megakaryocytosis. The effects of specifically targeting Lyn in human megakaryocytes and platelets, however, are not known. We hypothesized that conditionally disrupting Lyn during the process of iPSC to megakaryocyte differentiation might be an effective way to enhance megakaryocyte differentiation, platelet production, and hemostatic function. Using CRISPR/Cas9 gene editing technology, we generated a temporally controllable, tamoxifen-inducible Lyn-KO iPS cell line system that allowed us to conditionally delete Lyn at any desired stage of the iPSC→hematopoietic stem cell (HSC)→megakaryocyte→platelet differentiation process. We found tamoxifen efficiently disrupted Lyn expression at any of these stages within 24-48 hours after addition of the drug to the cell culture, and also improved the efficiency and yield of the iPSC→megakaryocyte→proplatelet differentiation process. Lyn-deficient human megakaryocytes prepared in this fashion exhibited enhanced reactivity, as reported by increased Erk phosphorylation in response to thrombopoietin stimulation, and by an improved ability to support hemostasis in human blood in vitro, as determined using thromboelastometry (ROTEM). Taken together, these data suggest that temporal disruption of Lyn kinase might represent an effective strategy for the engineering and manufacture of in vitro-derived platelets for both diagnostic and future therapeutic use. Disclosures Newman: NIH: Research Funding; Bloodworks Northwest: Membership on an entity's Board of Directors or advisory committees; Rallybio Corporation: Consultancy.