The FES Gene at the 15q26 Coronary-Artery-Disease Locus Inhibits Atherosclerosis.

BACKGROUND: Genome-wide association studies have discovered a link between genetic variants on human chromosome 15q26.1 and increased coronary artery disease (CAD) susceptibility; however, the underlying pathobiological mechanism is unclear. This genetic locus contains the FES (FES proto-oncogene, tyrosine kinase) gene encoding a cytoplasmic protein-tyrosine kinase involved in the regulation of cell behavior. We investigated the effect of the 15q26.1 variants on FES expression and whether FES plays a role in atherosclerosis. METHODS AND RESULTS: Analyses of isogenic monocytic cell lines generated by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that monocytes with an engineered 15q26.1 CAD risk genotype had reduced FES expression. Small-interfering-RNA-mediated knockdown of FES promoted migration of monocytes and vascular smooth muscle cells. A phosphoproteomics analysis showed that FES knockdown altered phosphorylation of a number of proteins known to regulate cell migration. Single-cell RNA-sequencing revealed that in human atherosclerotic plaques, cells that expressed FES were predominately monocytes/macrophages, although several other cell types including smooth muscle cells also expressed FES. There was an association between the 15q26.1 CAD risk genotype and greater numbers of monocytes/macrophage in human atherosclerotic plaques. An animal model study demonstrated that Fes knockout increased atherosclerotic plaque size and within-plaque content of monocytes/macrophages and smooth muscle cells, in apolipoprotein E-deficient mice fed a high fat diet. CONCLUSIONS: We provide substantial evidence that the CAD risk variants at the 15q26.1 locus reduce FES expression in monocytes and that FES depletion results in larger atherosclerotic plaques with more monocytes/macrophages and smooth muscle cells. This study is the first demonstration that FES plays a protective role against atherosclerosis and suggests that enhancing FES activity could be a potentially novel therapeutic approach for CAD intervention.

Neutrophils discriminate live from dead bacteria by integrating signals initiated by Fprs and TLRs.

The mechanisms whereby neutrophils respond differentially to live and dead organisms are unknown. We show here that neutrophils produce 5- to 30-fold higher levels of the Cxcl2 chemokine in response to live bacteria, compared with killed bacteria or isolated bacterial components, despite producing similar levels of Cxcl1 or pro-inflammatory cytokines. Secretion of high levels of Cxcl2, which potently activates neutrophils by an autocrine mechanism, requires three signals. The first two signals are provided by two different sets of signal peptides released by live bacteria, which selectively activate formylated peptide receptor 1 (Fpr1) and Fpr2, respectively. Signal 3 originates from Toll-like receptor activation by microbial components present in both live and killed bacteria. Mechanistically, these signaling pathways converge at the level of the p38 MAP kinase, leading to activation of the AP-1 transcription factor and to Cxcl2 induction. Collectively, our data demonstrate that the simultaneous presence of agonists for Fpr1, Fpr2, and Toll-like receptors represents a unique signature associated with viable bacteria, which is sensed by neutrophils and induces Cxcl2-dependent autocrine cell activation.

Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis.

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.

Identification of FES as a Novel Radiosensitizing Target in Human Cancers.

PURPOSE: The identification of novel targets for developing synergistic drug-radiation combinations would pave the way to overcome tumor radioresistance. We conducted cell-based screening of a human kinome siRNA library to identify a radiation-specific kinase that has a synergistic toxic effect with radiation upon inhibition and is not essential for cell survival in the absence of radiation. EXPERIMENTAL DESIGN: Unbiased RNAi screening was performed by transfecting A549 cells with a human kinome siRNA library followed by irradiation. Radiosensitizing effects of a target gene and involved mechanisms were examined. RESULTS: We identified the nonreceptor protein tyrosine kinase FES (FEline Sarcoma oncogene) as a radiosensitizing target. The expression of FES was increased in response to irradiation. Cell viability and clonogenic survival after irradiation were significantly decreased by FES knockdown in lung and pancreatic cancer cell lines. In contrast, FES depletion alone did not significantly affect cell proliferation without irradiation. An inducible RNAi mouse xenograft model verified in vivo radiosensitizing effects. FES-depleted cells showed increased apoptosis, DNA damage, G2-M phase arrest, and mitotic catastrophe after irradiation. FES depletion promoted radiation-induced reactive oxygen species formation, which resulted in phosphorylation of S6K and MDM2. The radiosensitizing effect of FES knockdown was partially reversed by inhibition of S6K activity. Consistent with the increase in phosphorylated MDM2, an increase in nuclear p53 levels was observed, which appears to contribute increased radiosensitivity of FES-depleted cells. CONCLUSIONS: We uncovered that inhibition of FES could be a potential strategy for inducing radiosensitization in cancer. Our results provide the basis for developing novel radiosensitizers.

Epigenomic profiling of DNA methylation for hepatocellular carcinoma diagnosis and prognosis prediction.

BACKGROUND AND AIM: DNA hypermethylation has emerged as a novel molecular biomarker for the diagnosis and prognosis prediction of many cancers. We aimed to identify clinically useful biomarkers regulated by DNA methylation in hepatocellular carcinoma (HCC). METHODS: Genome-wide methylation analysis in HCCs and paired noncancerous tissues was performed using an Illumina Infinium HumanMethylation 450K BeadChip array. Methylation-specific polymerase chain reaction and pyrosequencing were used to validate the methylation status of selected genes in 100 paired HCCs and noncancerous samples. RESULTS: A total of 97 027 (20.0%) out of 485 577 CpG sites significantly were differed between HCC and noncancerous tissues. Among all the significant CpG sites, 48.8% are hypermethylated and 51.2% are hypomethylated in HCCs. Multiple signaling pathways (AMP-activated protein kinase, estrogen, and adipocytokine) involved in gene methylation were identified in HCC. FES was selected for further analysis based on its high level of methylation confirmed by polymerase chain reaction and pyrosequencing. The result showed that FES hypermethylation was correlated with tumor size (0.001), serum alpha fetoprotein (0.023), and tumor differentiation (0.006). FES protein was significantly downregulated in 51/100 (51%) HCCs, and 94.12% (48/51) of them were due to promoter hypermethylation. Both FES hypermethylation and protein downregulation were associated with the progression-free survival and overall survival of HCC patients. Overexpressed and knockdown of FES confirmed its inhibitory effect on the proliferation and migration of HCC cells. CONCLUSIONS: We identified many new differentially methylated CpGs in HCCs and demonstrate that FES functions as a tumor suppressor gene in HCC and its methylation status could be used as an indicator for prognosis of HCC.

Whole transcriptome profiling of taste bud cells.

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

Comparative oncogenomics identifies tyrosine kinase FES as a tumor suppressor in melanoma.

Identification and functional validation of oncogenic drivers are essential steps toward advancing cancer precision medicine. Here, we have presented a comprehensive analysis of the somatic genomic landscape of the widely used BRAFV600E- and NRASQ61K-driven mouse models of melanoma. By integrating the data with publically available genomic, epigenomic, and transcriptomic information from human clinical samples, we confirmed the importance of several genes and pathways previously implicated in human melanoma, including the tumor-suppressor genes phosphatase and tensin homolog (PTEN), cyclin dependent kinase inhibitor 2A (CDKN2A), LKB1, and others. Importantly, this approach also identified additional putative melanoma drivers with prognostic and therapeutic relevance. Surprisingly, one of these genes encodes the tyrosine kinase FES. Whereas FES is highly expressed in normal human melanocytes, FES expression is strongly decreased in over 30% of human melanomas. This downregulation correlates with poor overall survival. Correspondingly, engineered deletion of Fes accelerated tumor progression in a BRAFV600E-driven mouse model of melanoma. Together, these data implicate FES as a driver of melanoma progression and demonstrate the potential of cross-species oncogenomic approaches combined with mouse modeling to uncover impactful mutations and oncogenic driver alleles with clinical importance in the treatment of human cancer.

MicroRNA-125b inhibits AML cells differentiation by directly targeting Fes.

MicroRNA-125b (miR-125b) has been reported to be upregulated in several kinds of leukemia, suggesting that miR-125b plays a role in Leukemia development. In this study, it was shown that miR-125b expression level decreased in response to 1α, 25-dihydroxy-vitamin D3 (1,25D3) in a dose- and time-dependent manner and miR-125b blocked 1,25D3-induced monocytic differentiation of U937 cells. In addition, miR-125b decreased mRNA expression of myelomonocytic differentiation markers, including CD11c, CD18 and CD64 and arrested the cell cycle at the S phase in U937 and HL60 cells. Fes was identified as a novel direct target of miR-125b and miR-125b could also reduce the expression levels of PU.1 and macrophage colony-stimulating factor receptor (MCSFR). Furthermore, Fes was found to be involved in monocytic differentiation via upregulation of PU.1 and MCSFR and Fes siRNA could also inhibit 1,25D3-induced monocytic differentiation of U937 and HL60 cells and decrease mRNA expression of CD11c, CD18 and CD64. Importantly, the inhibition of Fes siRNA on 1,25D3-induced monocytic differentiation could be rescued by transfection with miR-125b inhibitor. Our data highlights an important role of miR-125b in AML progression, implying the potential application of miR-125b in AML therapy.

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.

Syndapin--a membrane remodelling and endocytic F-BAR protein.

Syndapin [also called PACSIN (protein kinase C and casein kinase II interacting protein)] is an Fes-CIP4 homology Bin-amphiphysin-Rvs161/167 (F-BAR) and Src-homology 3 domain-containing protein. Three genes give rise to three main isoforms in mammalian cells. They each function in different endocytic and vesicle trafficking pathways and provide critical links between the cytoskeletal network in different cellular processes, such as neuronal morphogenesis and cell migration. The membrane remodelling activity of syndapin via its F-BAR domain and its interaction partners, such as dynamin and neural Wiskott-Aldrich syndrome protein binding to its Src-homology 3 domain, are important with respect to its function. Its various partner proteins provide insights into its mechanism of action, as well as its differential roles in these cellular processes. Signalling pathways leading to the regulation of syndapin function by phosphorylation are now contributing to our understanding of the broader functions of this family of proteins.

A new signaling pathway (JAK-Fes-phospholipase D) that is enhanced in highly proliferative breast cancer cells.

The products of the oncogene Fes and JAK3 are tyrosine kinases, whose expressions are elevated in tumor growth, angiogenesis, and metastasis. Phosphatidic acid, as synthesized by phospholipase D (PLD), enhances cancer cell survival. We report a new signaling pathway that integrates the two kinases with the lipase. A new JAK3-Fes-PLD2 axis is responsible for the highly proliferative phenotype of MDA-MB-231 breast cancer cells. Conversely, this pathway is maintained at a low rate of expression and activity levels in untransformed cells such as MCF10A. We also deciphered the inter-regulation that exists between the two kinases (JAK3 and the oncogene Fes) and between these two kinases and the lipase (PLD2). Whereas JAK3 and Fes marginally activate PLD2 in non-transformed cells, these kinases greatly enhance (>200%) PLD activity following protein-protein interaction through the SH2 domain and the Tyr-415 residue of PLD2. We also found that phosphatidic acid enhances Fes activity in MDA-MB-231 cells providing a positive activation loop between Fes and PLD2. In summary, the JAK3, Fes and PLD2 interactions in transformed cells maintain PLD2 at an enhanced level that leads to abnormal cell growth. Modulating this new JAK3-Fes-PLD2 pathway could be important to control the highly invasive phenotype of breast cancer cells.

FES kinase promotes mast cell recruitment to mammary tumors via the stem cell factor/KIT receptor signaling axis.

KIT receptor is required for mast cell development, survival, and migration toward its ligand stem cell factor (SCF). Many solid tumors express SCF and this leads to mast cell recruitment to tumors and release of mediators linked to tumor angiogenesis, growth, and metastasis. Here, we investigate whether FES protein-tyrosine kinase, a downstream effector of KIT signaling in mast cells, is required for migration of mast cells toward SCF-expressing mammary tumors. Using a novel agarose drop assay for chemotaxis of bone marrow-derived mast cells (BMMC) toward SCF, we found that defects in chemotaxis of fes-null BMMCs correlated with disorganized microtubule networks in polarized cells. FES displayed partial colocalization with microtubules in polarized BMMCs and has at least two direct microtubule binding sites within its N-terminal F-BAR and SH2 domains. An oligomerization-disrupting mutation within the Fer/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain had no effect on microtubule binding, whereas microtubule binding to the SH2 domain was dependent on the phosphotyrosine-binding pocket. FES involvement in mast cell recruitment to tumors was tested using the AC2M2 mouse mammary carcinoma model. These tumor cells expressed SCF and promoted BMMC recruitment in a KIT- and FES-dependent manner. Engraftment of AC2M2 orthotopic and subcutaneous tumors in control or fes-null mice, revealed a key role for FES in recruitment of mast cells to the tumor periphery. This may contribute to the reduced tumor growth and metastases observed in fes-null mice compared with control mice. Taken together, FES is a potential therapeutic target to limit the progression of tumors with stromal mast cell involvement.

Small-molecule inhibitors of the c-Fes protein-tyrosine kinase.

The c-Fes protein-tyrosine kinase modulates cellular signaling pathways governing differentiation, the innate immune response, and vasculogenesis. Here, we report the identification of types I and II kinase inhibitors with potent activity against c-Fes both in vitro and in cell-based assays. One of the most potent inhibitors is the previously described anaplastic lymphoma kinase inhibitor TAE684. The crystal structure of TAE684 in complex with the c-Fes SH2-kinase domain showed excellent shape complementarity with the ATP-binding pocket and a key role for the gatekeeper methionine in the inhibitory mechanism. TAE684 and two pyrazolopyrimidines with nanomolar potency against c-Fes in vitro were used to establish a role for this kinase in osteoclastogenesis, illustrating the value of these inhibitors as tool compounds to probe the diverse biological functions associated with this unique kinase.

FES/FER kinase signaling in hematopoietic cells and leukemias.

FES and FES-related (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors involved in regulating hematopoietic cell development, survival, migration, and inflammatory mediator release. Activated alleles of FES are potent inducers of myeloid differentiation, however FES-deficient mice have only subtle differences in hematopoiesis. This may reflect overlapping function of other kinases such as FER. Studies of FES- and FER-deficient mice have revealed more prominent roles in regulating the activation of mature innate immune cells, including macrophages and mast cells. Recently, new insights into regulation of FES/FER kinases has emerged with the characterization of their N-terminal phospholipid-binding and membrane targeting FER/CIP4 homology-Bin/Amphyphysin/Rvs (F-BAR) and F-BAR extension (FX) domains. The F-BAR/FX domains regulate subcellular localization and FES/FER kinase activation. FES kinase activity is also enhanced upon ligand binding to its SH2 domain, which may lead to further phosphorylation of the same ligand, or other ligand-associated proteins. In mast cells, SH2 ligands of FES/FER include KIT receptor PTK, and the high affinity IgE receptor (FceRI) that trigger rapid activation of FES/FER and signaling to regulators of the actin cytoskeleton and membrane trafficking. Recently, FES/FER have also been implicated in growth and survival signaling in leukemias driven by oncogenic KIT and FLT3 receptors. With further definition of their roles in immune cells and their progenitors, FES/FER may emerge as relevant therapeutic targets in inflammatory diseases and leukemias.

Miniaturized, microarray-based assays for chemical proteomic studies of protein function.

Systematic analysis of protein and enzyme function typically requires scale-up of protein expression and purification prior to assay development; this can often be limiting. Miniaturization of assays provides an alternative approach, but simple, generic methods are in short supply. Here we show how custom microarrays can be adapted to this purpose. We discuss the different routes to array fabrication and describe in detail one facile approach in which the purification and immobilization procedures are combined into a single step, significantly simplifying the array fabrication process. We illustrate this approach by reference to the creation of arrays of human protein kinases and of human cytochrome P450s. We discuss methods for both ligand-binding and turnover-based assays, as well as data analysis on such arrays.

Phospholipase D2 (PLD2) shortens the time required for myeloid leukemic cell differentiation: mechanism of action.

Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation→PLD2 overexpression→PLD2/Fes association→phosphatidic acid-led activation of Fes kinase→granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.

Role of the non-receptor tyrosine kinase fes in cancer.

Non receptor protein tyrosine kinases are targets in the treatment of a number of diseases. This review focuses on the role of Fes tyrosine kinase and on the design of inhibitors of this protein. Fes and its homologously related protein Fer are the only two members of a distinct class of non receptor tyrosine kinases and they seem to play a role in cytoskeletal rearrangements and inside-out signaling associated with receptor-ligand, cell-matrix and cell-cell interactions. The knowledge of the three dimensional structure of this protein, in fact, has informed drug design, while at the same time it has helped to shed some light on the molecular mechanism at the basis of kinase activation and functions.

Structure and regulation of the c-Fes protein-tyrosine kinase.

The c-Fes protein-tyrosine kinase is the normal cellular ortholog of several avian and feline retroviral oncoproteins. Unlike its transforming viral counterparts, c-Fes tyrosine kinase activity is tightly regulated in vivo through a mechanism involving coiled-coil oligomerization domains and other unique structural features found in its long N-terminal region. This review is focused on the regulatory features and structural biology of c-Fes, which has been implicated in normal cellular growth regulation, the innate immune response, and tumorigenesis.

Serotonin transporter, sex, and hypoxia: microarray analysis in the pulmonary arteries of mice identifies genes with relevance to human PAH.

Pulmonary arterial hypertension (PAH) is up to threefold more prevalent in women than men. Female mice overexpressing the serotonin transporter (SERT; SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH, whereas male SERT+ mice remain unaffected. To further investigate these sex differences, microarray analysis was performed in the pulmonary arteries of normoxic and chronically hypoxic female and male SERT+ mice. Quantitative RT-PCR analysis was employed for validation of the microarray data. In relevant groups, immunoblotting was performed for genes of interest (CEBPß, CYP1B1, and FOS). To translate clinical relevance to our findings, CEBPß, CYP1B1, and FOS mRNA and protein expression was assessed in pulmonary artery smooth muscle cells (PASMCs) derived from idiopathic PAH (IPAH) patients and controls. In female SERT+ mice, multiple pathways with relevance to PAH were altered. This was also observed in chronically hypoxic female SERT+ mice. We selected 10 genes of interest for qRT-PCR analysis (FOS, CEBPß, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1, and C1S), and 100% concordance was reported. Protein expression of three selected genes, CEBPß, CYP1B1, FOS, was also upregulated in female SERT+ mice. Serotonin and 17ß-estradiol increased CEBPß, CYP1B1, and FOS protein expression in PASMCs. In addition, CEBPß, CYP1B1, and FOS mRNA and protein expression was also increased in PASMCs derived from IPAH patients. Here, we have identified a number of genes that may predispose female SERT+ mice to PAH, and these findings may also be relevant to human PAH.