Fyn Expression Research Articles

Isoform-specific upregulation of FynT kinase expression is associated with tauopathy and glial activation in Alzheimer's disease and Lewy body dementias.

Cumulative data suggest the involvement of Fyn tyrosine kinase in Alzheimer's disease (AD). Previously, our group has shown increased immunoreactivities of the FynT isoform in AD neocortex (with no change in the alternatively spliced FynB isoform) which associated with neurofibrillary degeneration and reactive astrogliosis. Since both the aforementioned neuropathological features are also variably found in Lewy Body dementias (LBD), we investigated potential perturbations of Fyn expression in the post‐mortem neocortex of patients with AD, as well as those diagnosed as having one of the two main subgroups of LBD: Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB). We found selective upregulation of FynT expression in AD, PDD, and DLB which also correlated with cognitive impairment. Furthermore, increased FynT expression correlated with hallmark neuropathological lesions, soluble β‐amyloid, and phosphorylated tau, as well as markers of microglia and astrocyte activation. In line with the human post‐mortem studies, cortical FynT expression in aged mice transgenic for human P301S tau was upregulated and further correlated with accumulation of aggregated phosphorylated tau as well as with microglial and astrocytic markers. Our findings provide further evidence for the involvement of FynT in neurodegenerative dementias, likely via effects on tauopathy and neuroinflammation.

Tyrosine kinase Fyn promotes apoptosis after intracerebral hemorrhage in rats by activating Drp1 signaling.

Tyrosine kinase Fyn is a member of the Src kinase family, which is involved in neuroinflammation, apoptosis, and oxidative stress. Its role in intracerebral hemorrhage (ICH) is not fully understood. In this study, we found that Fyn was significantly elevated in human brain tissue after ICH. Accordingly, we investigated the role of Fyn in a rat ICH model, which was constructed by injecting blood into the right basal ganglia. In this model, Fyn expression was significantly upregulated in brain tissue adjacent to the hematoma. SiRNA-induced Fyn knockdown was neuroprotective for secondary cerebral damage, as demonstrated by reduced brain edema, suppression of the modified neurological severity score, and mitigation of blood-brain barrier permeability and neuronal damage. Fyn downregulation reduced apoptosis following ICH, as indicated by downregulation of apoptosis-related proteins AIF, Cyt.c, caspase 3, and Bax; upregulation of anti-apoptosis-related protein Bcl-2; and decreased tunnel staining. Mdivi-1, a Drp1 inhibitor, reversed Fyn overexpression induced pro-apoptosis. However, Fyn did not significantly affect inflammation-related proteins NF-κB, TNF-α, caspase 1, MPO, IL-1β, or IL-18 after ICH. Fyn activated Drp1 signaling by phosphorylating Drp1 at serine 616, which increased apoptosis after ICH in rats. This study clarifies the relationship between Fyn, apoptosis, and inflammation following ICH and provides a new strategy for exploring the prevention and treatment of ICH. KEY MESSAGES: ICH induced an increase in Fyn expression in human and rat cerebral tissues. Knockdown of Fyn prevented cerebral damage following ICH. Inhibition of Fyn had no significant effects on inflammatory responses. However, the downregulation of Fyn exerted neuroprotective effects on apoptosis. Fyn perturbed ICH-induced cell apoptosis by interacting with and phosphorylating (Ser616) Drp1 in a rat ICH model.

High Expression of the Tyrosine Kinase Fyn Causes Accelerated Differentiation of Growth Plate Chondrocytes in Young Rats with Chronic Renal Failure

Background: To observe the effect of high expression of the tyrosine kinase Fyn on chondrocyte differentiation in the tibial growth plate of young rats with chronic renal failure (CRF) and to explore the related molecular mechanism. Methods: The level of Fyn, the levels of the key Wnt/β-catenin pathway protein β-catenin and its phosphorylated form, p-β-catenin (Tyr142), and the level of the chondrocyte differentiation marker collagenX were observed in the CRF group in vivo and in vitro. The direct effect of Fyn and p-β-catenin in the cytoplasm of chondrocytes of rats with CRF was observed by using coimmunoprecipitation (co-IP). The levels of p-β-catenin (Tyr142), β-catenin and collagenX were observed after altering the Fyn levels in the chondrocytes of rats with CRF. Results: When CRF occurred in young rats, the level of Fyn in chondrocytes increased, the levels of p-β-catenin (Tyr142) and β-catenin in the Wnt/β-catenin pathway increased, and the level of the chondrocyte differentiation marker collagenX increased. The levels of p-β-catenin (Tyr142), β-catenin and collagenX were obviously increased when the Fyn level was increased in the chondrocytes of rats with CRF. In contrast, the levels of p-β-catenin (Tyr142), β-catenin and collagenX significantly decreased when the Fyn level was decreased. The Fyn protein and the p-β-catenin (Tyr142) protein directly interacted in the cytoplasm of the chondrocytes from the CRF group. Conclusion: The level of the tyrosine kinase Fyn in the tibial growth plate chondrocytes of young rats with CRF significantly increased, and the levels of the wnt/β-catenin pathway-related protein β-catenin and its phosphorylated form, p-β-catenin (Tyr142), significantly increased. Fyn could promote β-catenin phosphorylation (Tyr142) and cause it to be released from the α-catenin and cadherin complex and enter the cytoplasm of chondrocytes. p-β-catenin (Tyr142) could be transformed into β-catenin by dynamic equilibrium in the cytoplasm of chondrocytes, or p-β-catenin (Tyr142) could enter the nucleus of chondrocytes from the cytoplasm, thus activating the wnt/β-catenin pathway and accelerating the differentiation of chondrocytes. Funding: This work was supported by grants from the China Postdoctoral Science Foundation (2020M680910), Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (no.20210019),the Natural Science Foundation of Shanxi Province of China (no. 201701D121161) and the Key Research and Development Projects of Shanxi Province (no. 201803D31160). Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Ethics Committee of Shanxi Medical University [Approval number:SXMUE(2019004)] (Tai Yuan,China).

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

Profibrotic epithelial TGF-β1 signaling involves NOX4-mitochondria cross talk and redox-mediated activation of the tyrosine kinase FYN.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-β (TGF-β) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-β signaling is not known. Here, we demonstrate that TGF-β1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-β1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-β-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-β1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.

Fyn-tau Ablation Modifies PTZ-Induced Seizures and Post-seizure Hallmarks of Early Epileptogenesis.

Both Fyn and tau have been associated with neuronal hyperexcitability and neurotoxicity in many tauopathies, including Alzheimer's disease (AD). Individual genetic ablation of fyn or tau appears to be protective against aberrant excitatory neuronal activities in AD and epilepsy models. It is, however, still unknown whether ablation of both Fyn and tau can likely elicit more profound anti-seizure and neuroprotective effects. Here, we show the effects of genetic deletion of Fyn and/or tau on seizure severity in response to pentylenetetrazole (PTZ)-induced seizure in mouse models and neurobiological changes 24 h post-seizures. We used Fyn KO (fyn−/−), tau KO (tau−/−), double knockout (DKO) (fyn−/−/tau−/−), and wild-type (WT) mice of the same genetic background. Both tau KO and DKO showed a significant increase in latency to convulsive seizures and significantly decreased the severity of seizures post-PTZ. Although Fyn KO did not differ significantly from WT, in response to PTZ, Fyn KO still had 36 ± 8% seizure reduction and a 30% increase in seizure latency compared to WT. Surprisingly, in contrast to WT, Fyn KO mice showed higher mortality in <20 min of seizure induction; these mice had severe hydrocephalous. None of the tau−/− and DKO died during the study. In response to PTZ, all KO groups showed a significant reduction in neurodegeneration and gliosis, in contrast to WT, which showed increased neurodegeneration [especially, parvalbumin (PV)-GABAergic interneurons] and gliosis. DKO mice had the most reduced gliosis. Immunohistochemically, phospho-tau (AT8, pS199/S202), Fyn expression, as well as Fyn-tau interaction as measured by PLA increased in WT post-PTZ. Moreover, hippocampal Western blots revealed increased levels of AT8, tyrosine phospho-tau (pY18), and phosphorylated Src tyrosine family kinases (pSFK) in PTZ-treated WT, but not in KO, compared to respective controls. Furthermore, PV interneurons were protected from PTZ-induced seizure effects in all KO mice. The levels of inwardly rectifying potassium (Kir 4.1) channels were also downregulated in astrocytes in the WT post-PTZ, while its levels did not change in KO groups. Overall, our results demonstrated the role of Fyn and tau in seizures and their impact on the mediators of early epileptogenesis in PTZ model.

Chromatin accessibility mapping of the striatum identifies tyrosine kinase FYN as a therapeutic target for heroin use disorder

The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.

Expression Analysis of Fyn and Bat3 Signal Transduction Molecules in Patients with Chronic Lymphocytic Leukemia.

Background:Chronic lymphocytic leukemia (CLL) is correlated with defects in T-cell function resulting imparity in antitumor immune responses. Tim-3 is a co-inhibitory immune checkpoint receptor expressed on exhausted T-cells during tumor progression. Fyn and Bat3 are two important adaptor molecules involved in inhibition and activation of Tim-3 downstream signaling, respectively. In this study, the expression of Tim-3, Fyn, and Bat3 mRNA was evaluated in CLL patients. Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from 54 patients with CLL and 34 healthy controls. Total RNA was extracted from all samples and applied for cDNA synthesis. The relative expression of Tim-3, Fyn, and Bat3 mRNA was determined by TaqMan Real-Time PCR using GAPDH as an internal control. Results: Tim-3 mRNA expression was not significantly different between CLL patients and healthy controls. Fyn mRNA expression was significantly lower in CLL patients and conversely, Bat3 mRNA expression was higher in CLL patients compared to healthy controls. Interestingly, the mRNA expression of Fyn inhibitory adaptor molecule was remarkably associated with expression of Tim-3 in CLL patients. Conclusion:We have highlighted for the first time the expression of Fyn and Bat3 adaptor molecules in CLL patients. Our data demonstrated the strong correlation between the expression of Tim-3 and Fyn inhibitory molecules in CLL implying an important role for Tim-3-Fyn cooperation in induction of T-cell exhaustion.

FYN promotes gastric cancer metastasis by activating STAT3-mediated epithelial-mesenchymal transition.

Gastric cancer is one of the most lethal cancers worldwide. FYN, a gene that is differentially expressed in gastric cancer, is considered a critical metastasis regulator in several solid tumors, but its role in gastric cancer is still unclear. This study aimed to evaluate the role of FYN and test whether FYN promotes migration and invasion of gastric cancer cells in vitro and in vivo via STAT3 signaling. FYN was overexpressed in gastric cancer and positively correlated with metastasis. FYN knockdown significantly decreased cancer cell migration and invasion, whereas FYN overexpression increased cancer migration and invasion. Genetic inhibition of FYN decreased the number of metastatic lung nodules in vivo. Several epithelial-mesenchymal transition markers were positively correlated with FYN expression, indicative of FYN involvement in this transition. Furthermore, gene set enrichment analysis of a Cancer Genome Atlas dataset revealed that the STAT3 signaling pathway was positively correlated with FYN expression. STAT3 inhibition reversed the FYN-mediated epithelial-mesenchymal transition and suppressed metastasis. In conclusion, FYN promotes gastric cancer metastasis possibly by activating STAT3-mediated epithelial mesenchymal transition and may be a novel therapeutic target for gastric cancer.

FYN is required for ARHGEF16 to promote proliferation and migration in colon cancer cells

ARHGEF16 is a recently identified Rho-family guanine nucleotide exchange factor (GEF) that has been implicated in the activation of Rho-family GTPases such as Rho G, Rac, and Cdc42. However, its functions in colon cancer cell proliferation and migration are not well understood. In this study, we showed that ARHGEF16 was highly expressed in clinical specimens of colon cancer. In colon cancer cells, ARHGEF16-stimulated proliferation and migration in vitro and in vivo. Furthermore, we identified a nonreceptor tyrosine kinase, FYN, as a novel partner of ARHGEF16. Knocking down FYN expression decreased ARHGEF16 protein level in colon cancer cells. We further demonstrated that ARHGEF16-induced colon cancer cell proliferation and migration were dependent on FYN since knockdown FYN abolished the ARHGEF16-induced proliferation and migration of colon cancer cells. The FYN-ARHGEF16 axis mediates colon cancer progression and is a potential therapeutic target for colon cancer treatment.

675 The phosphorylation of CD147 by Fyn plays a critical role in melanoma cell growth and metastasis

CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a transmembrane glycoprotein that is highly expressed in tumor cells, particularly melanoma cells, and plays critical roles in tumor cell metastasis through the regulation of matrix metalloprotease (MMP) expression. Studies have demonstrated that CD147 could promote melanoma progression through post-translational modification, such as glycosylation. However, the role of phosphorylation of CD147 in melanoma remains unclear. In this study, we identified Fyn, a member of Src family that regulates diverse physiological processes, as a novel interacting protein of CD147. Our findings demonstrated that Fyn directly phosphorylates CD147 at Y140 and Y183. Two phosphospecific antibodies against Y140 and Y183 were developed to validate the phosphorylation of CD147 by Fyn. Moreover, the CD147-FF (Y140F/Y183F) mutation impaired the interaction between CD147 and GnT-V, leading to decreased CD147 glycosylation and membrane recruitment. In addition, CD147-FF significantly blocked MMP-9 expression as well as cell migration. Moreover, we found that Fyn is overexpressed in melanoma clinical tissues as well as in melanoma cell lines. Knockdown of Fyn expression markedly attenuated the malignant phenotype of melanoma cells in vitro and in vivo through downregulation of CD147 phosphorylation, indicating that Fyn/CD147 is a potential target molecule in melanoma treatment. Finally, through virtual screening, we identified amodiaquine as a potential inhibitor targeting the Fyn/CD147 axis. amodiaquine treatment dramatically inhibited the phosphorylation of CD147 by Fyn, thus attenuating melanoma cell growth and invasion in vitro and in vivo, suggesting that amodiaquine is a promising inhibitor for melanoma treatment. In conclusion, our findings showed that the Fyn/CD147 axis plays an essential role in melanoma cell metastasis and growth and that amodiaquine, which targets this axis, is a promising inhibitor for melanoma treatment.

The phosphorylation of CD147 by Fyn plays a critical role for melanoma cells growth and metastasis.

CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a transmembrane glycoprotein that is highly expressed in tumor cells, particularly melanoma cells, and plays critical roles in tumor cell metastasis through the regulation of matrix metalloprotease (MMP) expression. In this study, we identified Fyn as a novel interacting protein of CD147. Fyn is a member of the Src family of nonreceptor tyrosine kinases that regulates diverse physiological processes, such as T lymphocyte differentiation, through the TCR signaling pathway. Our findings demonstrated that Fyn directly phosphorylates CD147 at Y140 and Y183. Two phosphospecific antibodies against Y140 and Y183 were developed to validate the phosphorylation of CD147 by Fyn. Moreover, the CD147-FF (Y140F/Y183F) mutation impaired the interaction between CD147 and GnT-V, leading to decreased CD147 glycosylation and membrane recruitment. In addition, CD147-FF significantly blocked MMP-9 expression as well as cell migration. Moreover, we found that Fyn is overexpressed in clinical melanoma tissues as well as in melanoma cell lines. Knockdown of Fyn expression markedly attenuated the malignant phenotype of melanoma cells in vitro and in vivo through downregulation of CD147 phosphorylation, indicating that Fyn/CD147 is a potential target molecule in melanoma treatment. Finally, through virtual screening, we identified amodiaquine as a potential inhibitor targeting the Fyn/CD147 axis. Amodiaquine treatment dramatically inhibited the phosphorylation of CD147 by Fyn, thus attenuating melanoma cell growth and invasion in vitro and in vivo, suggesting that amodiaquine is a promising inhibitor for melanoma treatment.

Overexpression of FES might inhibit cell proliferation, migration, and invasion of osteosarcoma cells

BackgroundThis study aimed to screen osteosarcoma (OS) prognosis relevant genes for methylation dysregulation, and the functional mechanisms of FES overexpression in OS cells were investigated.MethodsThe OS prognosis relevant genes with differentially methylated positions (DMPs) identified from the GSE36001 and GSE36002 datasets, and the UCSC database, were used as a training set to construct a risk model, while the GSE21257 dataset was used as validation set. The expression levels of several key genes in OS cells after 5-Aza-2′-deoxycytidine treatment were detected by qPCR. The effects of FES overexpression on cell proliferation, cell cycle, migration, and invasion of MNNG/HOS were analyzed by CCK8, flow cytometry, and Transwell assays.ResultsA total of 31 candidate genes, corresponding to 36 DMPs, were identified as OS prognosis relevant genes; from these, the top 10 genes were used to construct a risk model. Following validation of the risk model, FES, LYL1, MAP4K1, RIPK3, SLC15A3, and STAT3 showed expression changes between the OS and control samples. qPCR results showed that the expression of FES was significantly downregulated in three OS cell lines and increased after 5-Aza-DC treatment. The proliferation, cell cycle progression, migration, and invasion of MNNG/HOS cells were significantly inhibited after transfection with FES overexpression plasmid, and the protein expression of FYN and β catenin were decreased in MNNG/HOS cells by FES overexpression.ConclusionsThe decrease in FES by hypermethylation was associated with OS prognosis, and might contribute to the proliferation, migration, and invasion of OS cells. FES, and its upstream FYN and β catenin, might coordinately exert a tumor suppressor effect in OS cells.

Overexpression of FYN suppresses the epithelial-to-mesenchymal transition through down-regulating PI3K/AKT pathway in lung adenocarcinoma

BackgroundTyrosine-protein kinase Fyn (FYN) plays a crucial role in Src family, which participates in the signal transduction of brain nerves and the development and activation of T lymphocytes in physiological conditions. We probed into the roles and mechanisms of FYN in lung adenocarcinoma (LUAD). MethodsCell activity, apoptosis, invasion, and migration were detected by CCK-8, FCM, transwell, and wound-healing assays, respectively. The angiogenesis capacity was evaluated by in vitro angiogenesis test. Relative mRNA and protein expressions were determined by qRT-PCR, Western blot, and immunohistochemistry assays, respectively. Insulin-like growth factors-I (IGF-I) was used as an agonist of PI3K/AKT pathway. ResultsWe demonstrated that FYN expression correlated with LUAD prognosis and was down-regulated in LUAD tissues and LUAD cells. Overexpression of FYN suppressed the cell viability, together with invasion and migration abilities of A549 cells. FYN overexpression accelerated the cell apoptosis and reduced the angiogenesis capacity of A549 cells. Overexpression of FYN suppressed E-cadherin, Vimentin, Snail, and PI3K/AKT expressions in A549 cells. High expression level of FYN reduced the migration and invasion capacities of A549 cells via down-regulating the PI3K/AKT pathway. ConclusionCollectively, our findings reveal that overexpression of FYN inhibits the epithelial-to-mesenchymal transition (EMT) through down-regulating the PI3K/AKT pathway in A549 cells.

MicroRNA-125a-3p participates in odontoblastic differentiation of dental pulp stem cells by targeting Fyn.

Fyn is a member of the protein tyrosine kinase family and its overexpression is associated with various types of inflammation. MicroRNAs can regulate the expression of target genes and play an important role in varied physiological and pathological processes. Based on the important role of Fyn and microRNA-125a-3p (miR-125a-3p) in inflammation, and combined with the bioinformatics studies, we performed in this study and chose miR-125a-3p as the focus of our research. During the progression of inflammation, we found that the expression of miR-125a-3p was decreased while the expression of Fyn was up-regulated. Fyn formed a complex with Neuropilin-1, which inhibited odontoblastic differentiation and expanded inflammatory responses through nuclear factor-κB signal pathways in dental pulp stem cells (DPSCs). These findings suggested that miR-125a-3p plays an important role in odontoblastic differentiation of DPSCs by targeting Fyn, implying its therapeutic potential in dental caries.

Fyn tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates antiglioma immune responses

High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn's role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion. We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8-/-, CD4-/-). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME. We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8-/- and CD4-/- immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME. Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.

Fyn stimulates the progression of pancreatic cancer via Fyn-GluN2b-AKT axis.

To assess the expression levels of Fyn in human tissue samples and pancreatic cancer cells and explore the potential mechanisms of Fyn in pancreatic cancer progression. Quantitative PCR and immunohistochemical (IHC) assays were performed to detect the expression of Fyn in 30 cancer tissue samples from pancreatic cancer patients and corresponding adjacent normal tissues. In addition, the potential correlations between Fyn expression levels and clinical pathological features were assessed. We further detected the effects of Fyn on the proliferation, apoptosis, migration, and invasion of the pancreatic cancer cells through colony formation assay, flow cytometry (FCM) assay, wound healing assay, and transwell assay, respectively. The potential effects of Fyn on tumor growth were assessed using an animal model. We demonstrated the possible involvement of Fyn in the progression of pancreatic cancer. We found that Fyn was upregulated in human pancreatic cancer tissues and cells, and we analyzed the correlations between Fyn expression and the clinicopathological features, including metastasis staging (p=0.010*) and tumor size (p=0.025*) of patients with pancreatic cancer. Our data further confirmed that Fyn affects cell proliferation, apoptosis, migration, and invasion of pancreatic cancer cells via the phosphorylation of GluN2b and regulation of AKT signaling pathway. We also demonstrated that Fyn promoted tumor growth of pancreatic cancer cells in vivo. We investigated the potential involvement of Fyn in the progression of pancreatic cancer, and therefore indicated Fyn as a possible therapeutic target for pancreatic cancer.

Fyn gene silencing reduces oligodendrocytes apoptosis through inhibiting ERK1/2 phosphorylation in epilepsy

This study aimed to investigate the effect of Fyn gene silencing on the apoptosis of oligodendrocytes (OLs) in epileptic model in vitro and the involved mechanism. Primary oligodendrocyte pro-genitor cells (OPCs) were separated from rats and differentiated to OLs. Immunofluorescent labeling showed positive expression of A2B5 in OPCs and Olig2 in OLs, suggesting the successful separation of OPCs and OLs. Three Fyn siRNAs (si-Fyn) and Fyn siRNA negative control (NC) were transfected into OLs. Western blot showed that among three si-Fyn groups, si-Fyn3 caused the lowest Fyn expression, so si-Fyn3 was chosen for following experiment. Cells were divided into four groups: Control, Model, NC and si-Fyn. In the Model group, cells were cultured in Mg-free extracellular fluid for 3 h. The morphology of control cells was normal. However, the migration of neurons, the aggregation of cell bodies and the “grid-like” changes of neural networks were observed in the model cells. OLs apoptosis in various groups was assessed by flow cytometry. Expression of Fyn, ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in OLs of various groups was evaluated by western blot. Compared with the Control group, the apoptotic rates, the Fyn expression and p-ERK1/2/ERK1/2 ratio in the Model and NC groups increased significantly (p < .05). However, the apoptotic rate, the Fyn expression and p-ERK1/2/ERK1/2 ratio in the si-Fyn group were remarkably smaller than those in the Model group (p < .05). In conclusion, Fyn gene silencing reduced the apoptosis of OLs through inhibiting the phosphorylation of ERK1/2 in epileptic model.

Cellular and Molecular Characterization of Breast Cancer Chemotherapy-Related B Cell Lymphomas

Chemotherapy can prolong life expectancy in patients with hematologic malignancies. However, the long-lasting effects of chemotherapy on the function of hematopoietic and immune systems remain to be fully characterized. The hematopoietic and immune systems are vital for lifelong supply of blood cells and defense against pathogens and malignancies. Cyclophosphamide (CTX) and Doxorubicin (DOX) are frequently used together as neoadjuvant and adjuvant therapy for breast cancer. The main goal of our project is to characterize in depth long-term adverse effects of CTX/DOX treatment on the function of hematopoietic and immune systems. A cohort of Ly5.2+ C57BL6 female mice were treated once a week for 5 consecutive weeks with 30 mg/kg CTX and 3 mg/kg DOX. Cohorts of age-matched control mice and CTX/DOX-treated mice were analyzed 15 months after the end of treatment. Comprehensive flow cytometry analysis of &gt;50 hematopoietic and immune cell types (HSPCs, B cells, T cells, myeloid, erythroid and megakaryocytic lineages) from the bone marrow (BM) and spleen (SPL) of individual age-matched control and CTX/DOX-treated mice, in combination with BM and SPL cell transplant from each mouse has revealed development of CTX/DOX-related B cell lymphoma (BCL) in 17% of CTX/DOX-treated mice. Moreover, 13% of the CTX/DOX-treated mice showed early stages of BCL development, while the remaining 70% of CTX/DOX-treated and all age-matched control mice did not show any signs of BCL. Chemo-treated mice developing BCL exhibited expansion of lymphoid-biased short-term HSCs (ST-HSC), significant reduction of Long-term HSCs (LT-HSC) in BM and SPL and increased population of immature B cell in the SPL. Transplant of BM and SPL cells from CTX/DOX-treated lymphoma-bearing (CTX/DOX-BCL) mice into Ly5.1+ syngeneic recipient mice resulted in massive splenomegaly, expansion of Ly5.2+ lymphoid-biased ST-HSCs and CLPs in the SPL, reduced Ly5.2+ myeloid-biased HSCs in BM and SPL and reduced Ly5.2+ MPPs and CMPs cells in the BM. Additionally, all Ly5.2+ B cell developmental stages were increased in CTX/DOX-BCL recipients. B1 cells were also significantly increased in the lymphoma transplant recipient mice, mimicking B1 cell overproduction in their donors. These results indicate that CTX/DOX treatment increases the incidence of cancer therapy-related B cell lymphoma development. Further immunophenotypic characterization of donor Ly5.2+ B cells in recipients identified coexistence of two BCL types: 1) CLL/SLL (CD19+ CD23+ CD20+ CD5+ B220-), and 2) Mantle Cell Lymphoma (MCL; CD19+ CD23- CD20+ CD5+ B220-), where MCL is predominant in BM and SPL. In contrast, mice transplanted with cells from CTX/DOX-treated "early stage lymphoma" mice did not have exorbitant amount of these two immunophenotypic populations in the SPL as compared to CTX/DOX-BCL recipient mice; but BM had a 2-fold increase of these cells compared to control. Likewise, transplant recipients of cells from "early stage lymphoma" and "normal" CTX/DOX donor mice had increased number of lymphoid-biased ST-HSCs in the SPL and reduced LT-HSCs in BM. Adding to the adverse effects of CTX/DOX on the immune system, populations of Natural Killer (NK) and Natural Killer T (NKT) cells were significantly decreased in recipients of cells from all CTX/DOX-treated mice compared to control transplant recipients. More importantly, RNAseq analysis revealed: 1) reduced expression of NK receptors (NKG2D, NKp46, CD94, CD69), 2) downregulated expression of PI3K, Fyn and PLCγ genes involved in NK cell-mediated cytotoxicity, and 3) upregulation of BAG6 and ULBP1 genes, known as suppressors of NK receptors, in CTX/DOX-BCL recipients compared to control recipients. Combined, these results suggest that attenuated numbers and impaired function of NK/NKT cells could be accountable for development of chemotherapy-related B cell lymphomas. Dysregulation of the hematopoietic system, expansion of BCL populations and attenuated NK/NKT function/surveillance in CTX/DOX-treated mice and mice transplanted with CTX/DOX-BCL cells demonstrate some of the harmful effects of CTX/DOX on the hematopoietic and immune system, causing the co-development of CLL and MCL B cell lymphomas. Thus, it is important to investigate direct effects of CTX/DOX on NK/NKT cells and determine the molecular and cellular causes and cellular origin (HSC, Ly-HSC, CLPs, B1 cells) of cancer therapy-related B cell lymphomas. Disclosures No relevant conflicts of interest to declare.

Targeting Neurotrophic Receptor Tyrosine Kinase (NTRK) Fusion Structural Homologs in Refractory Pediatric Acute Lymphoblastic Leukemia (ALL)

Introduction: Currently, the 5-year overall survival rate in relapsed pediatric ALL patients is only about 30%. Hence, new treatment approaches are urgently needed. Tyrosine receptor kinase (TRK) signalling pathways have been previously implicated in pediatric ALL relapse, although efficacious molecularly targeted therapies remain elusive. Larotrectinib is a recently approved inhibitor of Neurotrophic Receptor Tyrosine Kinase (NTRK) fusion proteins. Clinical trials have shown high efficacy of Larotrectinib, with limited side effects, in both adult and pediatric solid tumours. Here we show substantial homology between common NTRK fusion proteins and candidate members of the SRC family of TRKs, as well as high binding affinity of Larotrectinib to identified candidates and an impact on patient survival and prognosis. Our data provide the first evidence for a unique therapeutic approach for a subpopulation of refractory pediatric ALL. Methods: Using AutoDock Vina, molecular docking studies were conducted between Larotrectinib and multiple NTRK fusion structures to identify interaction regions with high affinity for Larotrectinib. Structural homologs with significant homology and coverage of substantial interaction regions were identified, and binding affinity to Larotrectinib was compared between the NTRK fusion structures and the identified homologs. To evaluate differential expression of these homologs, the mRNA expression of pediatric ALL bone marrow and normal bone marrow was collected from the GSE7186 dataset on the Gene Expression Omnibus (GEO). P-values of differential expression between ALL and normal mRNA expression of genes that encode for the identified homologs was determined. To evaluate the prognostic potential of the identified homologs in pediatric ALL, mRNA expression z-scores and overall survival data were collected from the recently published TARGET ALL dataset on cBioPortal. mRNA expression z-scores for the identified homologs were separated into quartiles, and survival of patients within the top and bottom quartiles were compared using the Kaplan-Meier survival analysis and the log-rank test for statistical significance. To evaluate the therapeutic potential of Larotrectinib, an inhibitor of both NTRK and the identified homologs was selected and its IC50 z-scores across cell lines and mRNA expression z-scores were analyzed from Genomics of Drug Sensitivity (GDSC) and Cancer Cell Line Encyclopedia (CCLE). IC50s were then compared between ALL and other cell lines. Spearman correlation values between the IC50s and mRNA expression of the identified homologs across all cell lines were verified to evaluate dose-dependency. Results: Four structural homologs were identified with significant homology and coverage with the NTRK fusion structures: FYN, YES1, FGR and SRC. Binding affinities to Larotrectinib for NTRK fusion structures and the identified homologs were not statistically significantly different. Differentially expressed genes in T-cell ALL were FYN (p=0.008), and YES1 (p=0.057) and in B-cell ALL was FYN (p=0.016). Kaplan-Meier survival analysis showed that poor prognosis was associated with low expression of FYN (p=0.006) and YES1 (p=0.000) and with high expression of FGR (p=0.068) and SRC (p=0.252). WZ3105 was identified as another inhibitor of both NTRK and the SRC kinase family, and IC50 of ALL cell lines were shown to be significantly more sensitive to this inhibitor than all other cell lines (p=0.016). Spearman analysis showed significant correlation between IC50s of WZ1305 and cell line mRNA expression of SRC (p=0.015) and FGR (p=0.027), indicating dose-dependency. Conclusion: In addition to NTRK fusion structures, Larotrectinib has been found to have substantial binding affinity with several members of the SRC kinase family. Among the homologs, both FYN and YES1 showed significant differential expression and prognostic significance in ALL. Interestingly, high expressions FGR and SRC are associated with poor prognosis in ALL, therefore representing potential targets. The Larotrectinib analog, WZ3105, is particularly efficacious in ALL cell lines, and dose-dependency with FGR and SRC expression. As such, we provide molecular bioinformatics and data from experimental studies for the potential therapeutic efficacy of Larotrectinib in pediatric ALL, as an inhibitor for NTRK fusion structural homologs, specifically FGR and SRC. Disclosures Narendran: Bayer: Honoraria, Other: CANTRK Advisory Board .