SEMA6A is a multifunctional transmembrane semaphorin protein that participates in various cellular processes, including axon guidance, cell migration, and cancer progression. However, the role of SEMA6A in clear cell renal cell carcinoma (ccRCC) is unclear. Based on high-throughput sequencing data, here we report that SEMA6A is a novel target gene of the VHL-HIF-2α axis and overexpressed in ccRCC. Chromatin immunoprecipitation and reporter assays revealed that HIF-2α directly activated SEMA6A transcription in hypoxic ccRCC cells. Wnt/β-catenin pathway activation is correlated with the expression of SEMA6A in ccRCC; the latter physically interacted with SEC62 and promoted ccRCC progression through SEC62-dependent β-catenin stabilization and activation. Depletion of SEMA6A impaired HIF-2α-induced Wnt/β-catenin pathway activation and led to defective ccRCC cell proliferation both in vitro and in vivo. SEMA6A overexpression promoted the malignant phenotypes of ccRCC, which was reversed by SEC62 depletion. Collectively, this study revealed a potential role for VHL-HIF-2α-SEMA6A-SEC62 axis in the activation of Wnt/β-catenin pathway. Thus, SEMA6A may act as a potential therapeutic target, especially in VHL-deficient ccRCC. Renal cell carcinoma (RCC) is the second leading cause of death in patients with urinary cancer worldwide, with a frequency of 1/30,000, accounting for 2–3% of all adult cancers . It is cVHL) in renal epithelial cells, which deregulates the hypoxia-inducible factors (HIFs), including HIF-1a and HIF-2α [VHL can form a classical E3 ubiquitin ligase complex with elonginB, elonginC, Cullin2, and Rbx1 [HIF-2α for degradation through the ubiquitin-proteasome pathway [HIF-2α are hydroxylated by prolyl hydroxylase, and the hydroxylated HIF-2α is then recognized by this E3 ubiquitin ligase complex, followed by sequential degradation [VHL failed to recognize HIF-2α under hypoxia, resulting in its accumulation [HIF-2α is associated with the constitutively expressed HIF-1β to form a heterodimer, which is then transferred to the nucleus. Subsequently, the expression of the downstream genes, vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) [VHL mutations are observed in ~70–80% of ccRCC [VHL E3 ubiquitin ligase complex cannot be formed, resulting in the stabilization of the HIF-2α protein irrespective of the cellular oxygen supply [HIF-2α, together with HIF-1β, causes the abnormal activation of the downstream oncogenes and promotes the occurrence and development of ccRCC [Interestingly, ccRCC is triggered by the biallelic inactivation of the tumor suppressor gene Von Hippel-Lindau  , is acti (CCND1) –19. VHL of ccRCC , and then supply . The staof ccRCC .HIF-2α silenced, and VHL overexpressed ccRCC cells was integrated, which facilitated, which in turn led to the identification of SEMA6A as a direct downstream target gene of the VHL-HIF-2α axis. Next, we revealed that SEMA6A is required for the integrity of the HIF-2α-β-catenin signaling pathway in ccRCC, and the genetic deletion of SEMA6A resulted in impaired proliferation ability in ccRCC cells by repressing SEC62-dependent β-catenin stabilization and activation.Herein, the transcriptional profiling from p < 0.05 and |logFC| > 1.2 were set as the cutoff criteria. Venn diagrams were drawn to obtain intersecting genes, and Gene Ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) pathway analyses were performed. The mutation frequencies and copy number alterations in the SEMA6A gene were observed by genomic analysis of renal clear cell carcinoma  through the cBioPortal online tool . The transcript levels of SEMA6A were obtained from the GEPIA database (http://gepia.cancer-pku.cn) between normal kidney tissue and KIRC. The expression of SEMA6A in different subtypes of kidney renal clear cell carcinoma (KIRC) was analyzed via the UALCAN online tool .GSE149005, GSE32297, GSE68417, and GSE79683 were downloaded from the gene expression omnibus (GEO) database on the NCBI website.  and cultured in an appropriate medium containing 10% fetal bovine serum  and supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml), in a 37 °C incubator with 5% CO4 cells were seeded in a 96-well plate and CCK-8 reagent was added to the wells; the absorbance was measured at 450 or 490 nm. For bromodeoxyuridine (BrdU) assay, the cells were seeded in a 96-well plate and incubated for 24 h. Then, the cell culture medium was discarded, the cell fixative solution was added to each well, and the cells were fixed, denatured, and incubated with anti-BrdU and TMB substrate. The absorbance was measured at 450 nm on a microplate reader. For colony formation assay, about 1000 cells were seeded in each well of a 12-well plate, and the seeded 12-well plate was cultured for about 2 weeks in a 5% CO2 and 37 °C incubator. Cells were washed with phosphate-buffered saline (PBS), fixed with paraformaldehyde for 15–30 min, and stained with 0.05% crystal violet or Giemsa to count the number of colonies. To assess the cell migration ability, 5 × 105 cells were seeded in 12-well plates for 24 h. When the cell density was about 80%, a 10-μl pipette tip was used to draw a line perpendicular to the bottom of each well, washed three times with PBS to remove the floating cells. Images were taken under the microscope at the same position at 0, 24, and 48 h after scratching, and the migration area was calculated using ImageJ software.For cell counting kit-8 (CCK-8) analysis, 5 × 10SEMA6A and SEC62 were uploaded onto the I-TASSER webserver, and the modeling was run with default settings. Five structure clusters were obtained as results for each protein, the final models were selected according to the overall conformations, and the C-scores were calculated by the server. The protein-protein docking was performed using the Cluspro online server. Ligplot software was used to analyze the binding force between the two proteins, and Pymol software was used for three-dimensional (3D) protein-binding conformation imaging.Full-length sequences of SEMA6A and SEC62 was assessed by GST-pull-down assay. Briefly, GST or GST-SEC62 was expressed in BL21 cells and purified using glutathione-Sepharose beads. Equivalent amounts of GST or GST fusion proteins were resuspended in reaction buffer  containing 0.2 mg/ml bovine serum albumin (BSA). Then, 1 mg FLAG-SEMA6A-transfected cell lysate was added to each mixture, followed by rotation at room temperature for 1 h. Subsequently, the beads were boiled in a sample buffer to elute the bound proteins. The final sample was separated by SDS-PAGE and detected by immunoblotting.The IP protocol has been described previously . BrieflyHIF-2α , anti-SEMA6A , anti-SEC62, and anti-β-Actin.Cells were harvested and lysed with SDS-containing lysis buffer . An equivalent amount of protein from each sample was separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk for 1 h, the membranes were incubated with the indicated primary antibodies at 4 °C overnight. After washing with Tris buffered saline Tween (TBST) buffer , the membrane was incubated with the secondary antibody at 25 °C for 1 h. Then, the immunoreactive bands were developed using enhanced chemiluminescence reagent with β-Actin as a loading control. The primary antibodies were as follows: anti-SEMA6A or HIF-2α were purchased from Sigma. The lentiviral particles were used to infect ccRCC cells and screened by puromycin for ~2 weeks. RNA was extracted from the samples using the RNeasy extraction kit and reverse transcribed into cDNA using the reverse transcription kit according to the manufacturer’s instructions. RT-PCR was performed using Light Cycler 480, and β-actin was used as an internal reference gene. The relative expression of the target gene was analyzed using the 2−ΔΔCt method.Lentiviral human shRNA plasmids targeting SEMA6A gene was amplified from the genomic DNA of A498 cells and cloned into the pGL4.15 vector . After transfection, the cells were harvested and lysed, and the luciferase activity was assayed using the Dual Luciferase Assay System (Promega). The luciferase activity was normalized to Renilla luciferase activity. For ChIP assay, cells were fixed with formaldehyde, and DNA was sheared to fragments at 100–500 bp by sonication. Then, the supernatant was incubated with antibodies against HIF-2α or normal serum IgG at 4 °C overnight.The TOP/FOP flash reporters were purchased from Beyotime. The promoter region of human 6 cells in 300 μl PBS were injected subcutaneously into the flanks of 6-week-old male nude mice. Tumors were measured every 7 days after injection, the tumor volume was calculated according to the formula (length × width2)/2, and the mice were sacrificed 4 weeks after inoculation. All animal experiments were approved by the Institutional Animal Ethics Committee of Jiangsu Ocean University, and animal care was in accordance with the institutional guidelines.For subcutaneous xenografts, 3 × 10t test or one-way analysis of variance using Tukey’s test. Statistical significance was expressed as *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.All experiments were repeated at least three times. Data were expressed as mean ± standard deviation (SD). The statistical analysis was performed using GraphPad Prism 9.0 software . The differences between the groups were calculated using either the Student’s HIF-2α in ccRCC, we first analyzed the transcriptional profiling of HIF-2α-regulated genes in ccRCC cells using a previously published RNA sequencing (RNA-seq) dataset (GSE149005). GSE149005 contains a transcriptional gene expression profile of 786-O cells treated with sgRNAs to HIF-2α or scrambled control. p < 0.05 and |logFC| > 1.2 were set as the cutoff criteria. A total of 118 genes were upregulated, and 144 genes were downregulated after HIF-2α was depleted  was detected in the promoter region of the human SEMA6A gene  promoter  samples. The main genetic changes included gene amplification  and mutations    database  Fig. . The copsed Fig. . Also, tues Fig. . Moreoveues Fig. . Consist17) Fig. . We alsoase Fig. . We thenase Fig. . Taken tSEMA6A in ccRCC, we suppressed SEMA6A expression in ccRCC cells. A498 cells were treated by lentiviral infections with five independent shRNAs against SEMA6A. Although all shRNAs could inhibit the expression of SEMA6A, #1 and #3 showed the best inhibitory effect  mice. p < 0.05 and |logFC| > 1.2 were set as the cutoff criteria, and the DEGs were exhibited in both heatmap (Fig. SEMA6A KO mice tissue (Fig. Wnt signaling pathway is significantly suppressed in SEMA6A KO mice tissue (Fig. SEMA6A mRNA expression levels, and conducted GSEA. Compared to the low SEMA6A expression group, the Wnt/β-catenin signaling pathway was significantly enriched in the high SEMA6A expression (Fig. SEMA6A was positively correlated with both FZD1 and Myc, two well-known downstream target genes of the Wnt/β-catenin signaling pathway (Fig. SEMA6A could regulate the Wnt/β-catenin signaling pathway. Thus, we conducted a TOPFlash/FOPFlash assay to assess whether SEMA6A affects the transcriptional activity of β-catenin/TCF complex. We also found that the ectopic expression of SEMA6A significantly increases TOPflash activity, but not FOPflash activity, compared to control vector expression (Fig. SEMA6A decreased the TOPflash activity without affecting the FOPflash activity (Fig. CCND1 and C-Myc (Fig. β-catenin was unchanged in SEMA6A-depleted cells (Fig. SEMA6A expression decreased the protein levels of both β-catenin and CCND1 (Fig. SEMA6A regulates the stability of β-catenin. In agreement, both the proteasome inhibitor MG132 and the pan-cullin inhibitor MLN4924 prevented the decline of β-catenin protein levels in SEMA6A-depleted cells (Fig. SEMA6A increased the ubiquitination of β-catenin protein (Fig. SEMA6A activates the Wnt/β-catenin signaling pathway by promoting the stability of β-catenin protein. Typically, the administration of ICG-001, a small molecule compound that selectively antagonizes β-catenin/TCF-mediated transcriptional activity, significantly reversed SEMA6A-induced TOPflash activation (Fig. SEMA6A-induced A498 cell proliferation (Supplementary Fig. SEMA6A-regualted β-catenin activation is related to ccRCC progression. Consistent with previous findings, HIF-2α depletion significantly reduced the transcriptional activity of β-catenin/TCF complex, which could be partially restored by SEMA6A overexpression (Fig. SEMA6A mediates HIF-2α-induced activation of the Wnt/β-catenin signaling pathway. In conclusion, the current results indicated that SEMA6A activates the Wnt/β-catenin signaling pathway in ccRCC.To further determine the mechanism of action of map Fig.  and volcmap Fig. . Comparesue Fig. . Then, wion Fig. . In agreway Fig. . These dion Fig. . Conversity Fig. . The decMyc Fig. . Surprislls Fig. , while tND1 Fig. , suggestlls Fig. , indicatein Fig. . Togetheion Fig. . In addiion Fig. , indicatSEMA6A regulates the stability of β-catenin protein, we searched for SEMA6A-interacting protein. A total of 82 putative SEMA6A-interacting proteins were identified using the BioGRID dataset (Supplementary Fig. SEC62 formed a protein complex with β-catenin and inhibited the degradation of β-catenin protein [SEC62 is a binding partner of SEMA6A, we carried out structure-based computational analyses to show the interaction between SEMA6A and SEC62. Since the crystal structures for these two proteins are currently not available, we used the I-TASSER webserver for structural modeling. The protein-protein docking was performed using the Cluspro online server, a rigid protein docking algorithm based on a fast Fourier transform. Ligplot software was used to analyze the binding force between the two proteins, and Pymol software was used for 3D protein-binding conformation imaging. The modeling results revealed that many hydrogen bonding forces were formed between both proteins (Fig. SEMA6A might form a tight protein complex with SEC62. Subsequently, we purified endogenous SEMA6A protein from A498 cell lysate and detected the presence of SEC62 protein (Fig. SEC62 antibody (Fig. SEC62 and GST as baits and found that GST-tagged SEC62, but not GST alone, efficiently pulled down SEMA6A protein, suggesting a direct interaction between SEC62 and SEMA6A (Fig. SEC62 has competitively disrupted the interaction between β-catenin and APC to inhibit the β-catenin destruction complex assembly, driving us to assess whether SEMA6A affected the interaction between SEC62 and β-catenin. To test this hypothesis, we purified the SEC62 protein complex from A498 cells with or without SEMA6A depletion and then detected the presence of β-catenin by immunoblotting. Consistent with previous findings, SEC62 was bound to β-catenin in A498 cells. However, in the absence of SEMA6A, the interaction between SEC62 and β-catenin was impaired (Fig. SEMA6A significantly enhanced this interaction (Fig. SEMA6A promoted the interaction between SEC62 and β-catenin to prevent the degradation of β-catenin protein, thereby indicating that SEC62 might be critical for SEMA6A-induced β-catenin activation and biological functions. Luciferase assay showed that SEC62 knockdown decreased the transcriptional ability of β-catenin induced by SEMA6A (Fig. SEC62 depletion significantly reversed SEMA6A-induced A498 cell proliferation (Fig. SEC62 depletion also significantly reversed SEMA6A-induced A498 cell proliferation in nude mice in vivo (Fig. HIF-2α-induced SEMA6A expression promoted ccRCC progression through SEC62-dependent β-catenin stabilization and activation (Fig. To further investigate how  protein . To evalins Fig. , suggestein Fig. . Moreoveody Fig. . To deteA6A Fig. . Reportered Fig. . On the ion Fig. . TogetheA6A Fig. . Furtherion Fig. , BrdU inion Fig. , Caspaseion Fig. , colony ion Fig.  and invaion Fig. . Importaivo Fig. . Togetheion Fig. .Fig. 6SEVHL-HIF-2α axis based on the available transcriptome data. Next, we hypothesized that the mRNA levels of potential substrates of the VHL-HIF-2α axis are decreased in VHL-overexpressing cells and increased in HIF-2α-deficient cells. Moreover, the mRNA levels of these potential substrates are positively correlated with the activity of the HIF-2α signaling pathway. According to these screening criteria, 4 genes were identified, including SEMA6A and 3 known canonical target genes of HIF-2α. SEMA6A is a multifunctional protein that participates in various cellular processes, including vascular development and angiogenesis [SEMA6A can form a complex with plexins to regulate the actin cytoskeleton, motility, and cell proliferation and is involved in cancer development [SEMA6A was elevated under physical hypoxia or pharmacologically-simulated hypoxia conditions, whereas genetic ablation or pharmacological inhibition of HIF-2α significantly decreased SEMA6A expression in ccRCC cells, suggesting that hypoxia-induced SEMA6A expression requires the transcriptional activity of HIF-2α. Typically, ChIP and luciferase assays revealed that HIF-2α directly activates SEMA6A transcription in hypoxic ccRCC cells. The pooled TCGA and GEO data showed that SEMA6A mRNA was significantly increased in ccRCC compared to normal tissues and benign kidney tissues, suggesting that SEMA6A is involved in ccRCC progression. Consistent with this finding, depletion of SEMA6A significantly restrained the proliferation and migration ability of ccRCC in vitro and limited the in vivo growth of cancer in nude mice. Together, these results highlighted an oncogenic role of SEMA6A in ccRCC.In the present study, we screened the substrates of the ogenesis . SEMA6A elopment . Next, wSEMA6A KO mice tissue showed that the Wnt/β-catenin pathway is significantly downregulated in case of SEMA6A depletion. TCGA data analysis demonstrated that the mRNA expression of SEMA6A is closely correlated to the activation of Wnt/β-catenin pathway in ccRCC. The Wnt/β-catenin pathway is implicated in numerous signaling pathways, including embryogenesis, cell proliferation, migration and invasion, apoptosis, and organogenesis [SEMA6A decreases the Wnt/β-catenin activity, as shown by impaired TOPflash luciferase activity and reduced Wnt/β-catenin downstream target gene expression. Conversely, the overexpression of SEMA6A activated the Wnt/β-catenin pathway, which could be reversed by pharmacological β-catenin inhibition. Also, β-catenin is a multifunctional protein encoded by the CTNNB1 gene that mediates signal transduction and exerts a core role in promoting tumor proliferation and metastasis in various malignancies [SEMA6A knockdown inhibited the activity of the Wnt/β-catenin pathway, the mRNA level of β-catenin was not affected. Interestingly, the protein level of β-catenin is downregulated in SEMA6A-depleted cells, suggesting that SEMA6A regulates β-catenin at the posttranscriptional level. Consistent with this phenomenon, we showed that decreased β-catenin protein level in SEMA6A-depleted cells was caused by enhanced ubiquitination and degradation. The turnover of β-catenin is critical for the inactivation of the Wnt/β-catenin signaling pathway [SEC62, a component of the protein translocation tool on the endoplasmic reticulum membrane, competitively disrupted the interaction between β-catenin and APC to prevent β-catenin destruction [SEC62 is found to be a putative interaction protein of SEMA6A. Several technical methods at different levels confirmed the interaction between SEMA6A and SEC62 and found a critical role of SEMA6A in SEC62-mediated β-catenin stabilization and activation. Thus, our finding elucidated the molecular mechanism through which SEMA6A stabilizes β-catenin. We also found that depletion of SEC62 significantly reverses SEMA6A-induced malignant phenotypes in ccRCC both in vitro and in vivo, indicating a critical role of the SEMA6A-SEC62 axis in maintaining the malignant phenotypes of ccRCC.The transcriptome data analysis of ogenesis . The abnogenesis . The curgnancies . Althoug pathway . Reportetruction . InteresSEMA6A is identified as a novel target gene of VHL-HIF-2α with a cancer-promoting role in ccRCC. SEMA6A activates the Wnt/β-catenin signaling pathway by inhibiting the β-catenin ubiquitination and degradation, suggesting that SEMA6A acts as a potential therapeutic target in ccRCCs.Collectively, Supplementary Figure legendsSupplementary Fig 1Supplementary Fig 2Supplementary Fig 3Supplementary Fig 4Supplementary Fig 5Supplementary Fig 6Supplementary Table1Supplementary Table2Supplementary Table3Supplementary Table4Supplementary Table5Supplementary Table6Supplementary Table7Supplementary Table8Original Data FileReproducibility checklist


Type 2 diabetes mellitus (T2DM) is a chronic metabolic abnormality leading to microvascular and macrovascular complications. Non-insulin Incretin mimic synthetic peptide exendin-4 was introduced as an anti-diabetic drug which helped diabetic patients with triggering insulin secretion; further researches have revealed an effective role of exendin-4 in treatment of T2DM related diseases. Exendin-4 is approximately similar to Glucagon-like peptide, thus it can bind to the glucagon-like peptide-1 receptor (GLP-1R) and activated different signaling pathways that are involved in various bioactivities such as apoptosis, insulin secretion and inactivation of microglial. In this review, we investigated the interesting role of exendin-4 in various kinds of T2DM related disorders through the activation of different signaling pathways. T2DM is mainly identified by a hyperglycemic condition that is caused by the combination of cell resistance to insulin and/or insufficiency of pancreatic β cell to synthesize and release insulin to the systemic circulation in response to high levels of glucose. Irrespective of diabetic conditions, the imbalance of insulin could promote microvascular and macrovascular pathologies. The use of traditional approaches such as metformin administration and insulin therapy are the most common and prominent options for patients with T2DM  is a progressive metabolic disorder with a high rate of prevalence worldwide contributing to profound socio-economic implications.ith T2DM .3,4 Hoith T2DM . In the  monster .7,8 GLP-1 has the potential to reduce systemic glucose levels by inducing insulin secretion. This factor also delays gastric emptying, inhibits glucagon secretion and reduces food intake rate. GLP-1 exerts its effects through GLP-1R. GLP-1R is a G protein-coupled receptor from glucagon receptor family by located on chromosome 6. Several studies have confirmed that GLP-1R is not exclusive for pancreatic tissue but also seen in the peripheral tissues such as lungs, stomach, intestines, kidneys, and heart in addition to the most areas of the brain. This receptor contains two major domains ECD (extracellular domain) and TM (transmembrane domain). As previously documented in different experiments, it demonstrated that EX-4 regulates glucose level in diabetic subjects via triggering insulin secretion, reducing glucagon secretion and food intake. Compared to GLP-1, EX-4 is resistant to dipeptidyl peptidase-IV activity, thereby possess a much longer plasma half-life and activity.GLP-1 is formed by the post-translational stage of pro-glucagon hormone in the L-cells of the small intestine in response to the ingestion of food. However, there are some controversies related to EX-4 and GLP-1 interaction with GLP-1R. For instance, de Maturana et al showed the 400-fold binding affinity of Ex-4 (Kd = 6 nM) to the N-terminal domain of GLP-1R compared to GLP-1-GLP-1R attachment.In spite of the similarity between EX-4 and GLP-1 function and structure, there are some differences in the binding capacity to GLP-1R. The isolated GLP-1R ECD binds EX-4 with high affinity rather than GLP-1. Even, GLP-1 could attach with high affinity to TM part of GLP-1R with site-directed mutagenesis as compared with EX-4. In the case with GLP-1 N-terminal truncation, EX-4 loses affinity to attach this receptor but the interaction of GLP-1 with GLP-1 remains unaffected. As previously mentioned, different biological processes such as cell proliferation and apoptosis modulation are initiated through activation of phosphatidylinositol 3-kinase/Akt (PI3K/AKT) adenylyl cyclase/protein kinase A signaling pathways. In PI3K/AKT axis, the activation of PI3K phosphorylates Akt factor which in turn affects the dynamic of different factors. For example, it was mentioned that activated Akt could increase the level of nitric oxide (NO) by phosphorylation of endothelial nitric oxide (eNOS) and contributes to the modulation of nitrosative stress. Of note, EX-4 has the potential to regulate the function of neurons by the suppression of glycogen synthase kinase-3β (GSK-3β). The decrease of GSK-3β function declines Tau protein phosphorylation thereby prohibits the aggregation of α-synuclein. Therefore, EX-4 is able to modulate the function of neurons and endothelial cells (ECs) during pathological conditions. It is noteworthy to mention that the application of EX-4 could decrease the cytopathic effects of Parkinson’s and Alzheimer’s diseases. In addition, it was well-established that the activation of microglial nuclear factor-ƙB (NF-ƙB) after administration of EX-4 provokes pro-inflammatory responses. One of the possible therapeutic effects of EX-4 on the injured cells could be related to the modulation of apoptosis. It was determined that the activation of Akt by EX-4 inhibits Caspase cascade such as caspase-9 and -3. In addition, the inhibition of pro-apoptotic factors such as Bad and Forkhead box O (FOXO) transcription factors decreased apoptotic changes. The above-mentioned factors are thought to participate in different cell activities , IL-1 and -6, tumor necrosis factor-α (TNF-α)) and other cytokines participate actively in epithelium repair. Systemic and topical administration of Ex-4 was found to decrease superoxide anions content and serum level of IL-6. Ex-4 has also angiogenesis potential in diabetic subjects. For example, it was found that the CD34+/KDR+ positive endothelial progenitor cells in the rat model of diabetes. Based on the data, the tubulogenesis rate increased in Ex-4-treated cells. In line with these finding, protein levels of VEGFR-2, matrix metalloproteinase-2 (MMP-2), p-eNOS, and TGF-β in these cells showing an increased angiogenesis rate. In addition to angiogenesis potential, keratinocyte proliferation rate was also induced in Ex-4 treated rats. In gastro-intestinal tract, the frequency of gastric ulcer is also diminished after administration of Ex-4. The suppression of inflammation rate and oxidative stress is prohibited in the periphery of ulcers. These features are found to confine the progression of ulcers and accelerate the healing.The accumulation of pro-oxidants and byproducts in squamous and non-squamous epithelial layers leads to cutaneous and gastric wounds in diabetic subjects. Previous data demonstrated the anti-cancer properties of Ex-4 on cancer cell types such as breast cancer cells. As previously described, Ex-4 has the potential to inhibit breast cancer cell proliferation and metastasis and promote apoptosis by the modulation of effectors such as Caspase-9, Akt, and MMP-2. In addition, the clonogenic property of cancer cells by up-regulation of tissue inhibitor of metalloproteinases-1 and -2 expressions. It was elucidated that the exposure of tumor cells to Ex-4 has the potential to confine tumor mass via engaging GLP-1R signaling pathway. Moreover, Ex-4 has the potential to attenuate ovarian cancer cell proliferation with the induction of GLP-1R mRNA expression. The promotion of pro-inflammatory status governed by NF-κB seems to be a critical tumoricidal mechanism. In other conditions such as pancreas and prostate cancers, the size of tumors decreases after exposure to Ex-4. Ex-4 provokes CD8+ cytotoxic T cells and modulates the function of Foxp3+ regulatory T cells. These changes limit the expansion and progression of cancers. Based on data, the modulatory effect of Ex-4 is different regarding cancer type. For example, the cytotoxic effect of Ex-4 on prostate cancer is mediated by modulation of ERK-MAPK via the cAMP-PKA without affecting apoptotic signaling pathways. Therefore, cautions must be considered in cancer inhibition based on cancer phenotype. Insulin resistance is touted as one of the most metabolic disorders during the onset of cancers that lead to cachectic status. Previous studies noted that hyperinsulinemia in response to insulin resistance may lead to tumor expansion by increasing the hepatic IGF-1 level while protein content of insulin-like growth factor binding proteins 1 and 2 decreases. Irrespective of the reasons for induction of insulin production, high levels of insulin provokes estrogen bioactivity which in turn increases the risk of cancer formation in breast. It is suggested to apply a combined therapy for the better tumoricidal effect of Ex-4 in cancer candidates. Simultaneous application of metformin and Ex-4 was found to yield better therapeutic effects. Interestingly; Ex-4 sensitizes cancer cells to ionizing radiation through adenosine monophosphate activated protein kinase (AMPK) activation, contributing to the reduction of colony number and density via reduction of phosphorylated mTOR. The application of Ex-4 in endometrial cancer Ishikawa xenografts in nude mice yielded promising outcomes. Honors and Kinzig found the therapeutic effects of Ex-4 in preventing cancer-associated cachexia in male rats with Yoshida sarcoma. However, it seems that the Ex-4 modulatory effect was more prominent in cases with small-sized tumor masses compared to subjects with large sized cancers. Administration of Ex-4 is able to return insulin to basal levels in tumor-bearing candidates. These data stand for a fact that Ex-4 could be used as anti-cancer agent in diabetic subjects susceptible to incidence of different cancer types.T2DM has been shown to predispose the risk of many malignancies, such as colon and breast and other cancer types. In addition to neuroprotective potential of Ex-4 on CNS by the inhibition of apoptosis, this factor is also able to pass across the blood-brain barrier and binds to GLP-1R followed by the activation of adenylyl cyclase, PKC and mitogen-activated protein signaling pathways could regulate brain bioactivity and memory function. The administration of Ex-4 was shown to promote SERCA expression through activation of PKA/cAMP signaling pathways and subsequently leads to inhibition apoptosis after the onset of spinal cord injury. The levels of pro-apoptotic effectors such as Bax, Caspase-3 and cytochrome C are decreased while the expression of Bcl-2. Therefore, it seems that the application of Ex-4 could reverse the detrimental effects of Ex-4 on acute CNS pathologies. Based on data, the onset of diabetic changes could predispose the possibility of degenerative diseases such as Alzheimer’s disease or Parkinson’s disease. Under the emergence of degenerative diseases, hyperinsulinemia and down-regulated insulin signaling per se are seen routinely in the context of CNS. The promotion of degenerative diseases contributes to the unusual aggregation of peptides and/or proteins in the specific regions of the brain. For instance, the deposition of Aβ forms plaques in the extraneuronal area as well as Tau deposited in the form of filaments inside neurons. The introduction of insulin causes to activation of PI3K/AKT and GSK-3β, as two main downstream players in the context of the brain. Ex-4 has the potential to decrease the phosphorylation of GSK-3β and thereby inhibits the bioactivity of tau kinase. Along with these changes, the aggregation of Tau protein is dramatically diminished followed by extracellular protein accumulation removal. Most notably, the administration of Ex-4 could directly increase insulin signaling pathways via phosphorylation of insulin receptor substrate 1 (IRS-1). The phosphorylation of IRS-1 phosphorylation per se increased phosphorylated levels of AKT and GSK-3β contributing to the dephosphorylation of protein tau. The application of Ex-4 in experimentally induced Parkinson’s disease by using 1-methyl-4- phenyl-1, 2, 3, 6-tetrahydropyridine in the model of a mouse, decreased the degeneration of dopaminergic neurons and secretion of MMP-3 released from microglia. The occurrence of ischemic changes and stroke has been reported in diabetic candidates after the endothelial cells’ injury. It seems that the application of Ex-4 in diabetic changes could inhibit endothelial injury by engaging the PI3K/p-Akt/Bcl-xl/Bcl-2 axis.The existence of receptor GLP-1R has been previously described in CNS. Hyperglycemia has detrimental effects on cardiac structure and function thereby contributing to pathological cardiomyopathy, cardiac hypertrophy, interstitial fibrosis, as well as increased apoptosis and oxidative stress also reported after the onset of insulin resistance. The participation of distinct signaling pathways was reported in diabetic hearts. For instance, it has recently documented that the modulation of AMPK in the pathophysiology of cardiomyocytes metabolism. Therefore, pharmacological activation/inhibition of effector AMPK brings inevitable impacts on the status and intensity of cardiac injury exposed to various metabolic situations. In downstream pathways, AMPK affects the activity of the mTOR signaling pathway that acts as a cross-talk linker between different cell bioactivities. Similar to some mTOR blockers such as rapamycin, activation of GLP-1/GLP-1R axis phosphorylates AMPK and reduces hypertrophic stress. During the occurrence of myocardial infarction (MI), the reduction of Akt-1 and MAPK kinase-3 phosphorylation is seen in the infarcted zones. Ex-4 treatment was found to increase the phosphorylation of these factors and reduce the extent of aberrant remodeling and the number of apoptotic cells juxtaposed to infarct regions. Furthermore, Ex-4 has the potential to promote angiogenesis and neovascularization by the proliferation of vascular endothelial cells and α-smooth muscle actin positive cells toward injured cardiac tissue. In addition, it was shown that Ex-4 exerts a prophylactic role against myocardial injury via the activation of the Sirt1/PGC1α signaling pathway. The activation of this axis could promote mitochondrial function coincided with the suppression of oxidative stress insults. APPL1 plays versatile roles to inhibit cardiomyocytes apoptosis via the interaction with systemic adipokines, adiponectin. This interaction per se contributes to the promotion of AMPK and proliferator-activated receptor-α (PPAR-α). Under the occurrence if MI, Ex-4 decreases cardiac tissue apoptosis by amplifying the systemic adiponectin concentration and APPL1 activation and phosphorylation of AMPK. Other cardiovascular abnormalities mainly heart failure happens due to the disruption in calcium recycling. Ex-4 ameliorates Ca2+ homeostasis in HF subjects by promoting eNOS/cGMP/PKG axis and of restoration of SERCA2a activity. These changes reduce cytoplasmic Ca2+ content and activity of CaMKII.It is mighty that diabetic patients are susceptible to cardiovascular disorders. Along with these changes, the dynamic of multiple factors mainly angiotensins are changed. Of note, angiotensin-II is the main peptide that part take in the progression of atherosclerotic pathologies by the induction of VSMCs proliferation and migration via controlling the enzymes catalyze the phosphorylation of extracellular signal-regulated kinase 1/2 and JNK. Scientific literature added a notion that Ex-4 administration inhibits Ang II-induced phosphorylation of ERK1/2 and JNK, improving hypertension and atherosclerosis in the candidate population. In addition to critical role of ERK1/2 and JNK in the dynamic growth of VSMCs, some studies have revealed that nuclear receptor superfamily like neuron derived orphan receptor 1 (NOR1), one of the key regulators of VSMCs proliferation during the occurrence of atherosclerosis, activity removal may lead to the control of neo-intima formation in injured vascular context. The incubation of VSMCs with Ex-4 precisely regulates the proliferation of these cells through inhibiting NOR1 promoter activity. In patients with pulmonary arterial hypertension (PAH), a serious disorder which affects near to 30% of patients suffering from congenital heart disease is originated from extreme pulmonary blood flow due to excessive drug use, a genetic mutation in different factors like bone morphogenetic protein receptor type II. By applying Ex-4, the improvement of PAH is initiated via the reduction of pro-inflammatory cytokines interleukin-1α and 1β in PAH rats. After incubation of PAH rats with Ex-4, the function of right ventricular function was restored the number of the amount of Smooth muscle myosin heavy chain class II and α-SMA.The uncontrolled proliferation and migration of vascular smooth muscle cells (VSMCs) are touted as one of the risk factors resulting in atherosclerosis. During the development of atherosclerotic plaques, VSMCs undergo phenotype shifting with an enhanced proliferation rate. The promotion of diabetic nephropathy causes to chronic kidney disease by time, leading to renal insufficiency. The possibility of infectious nephritis is also reported in diabetic conditions. Commensurate with these data, lowering systemic glucose contents seems a priority in the control of nephropathy. The application of Ex-4 in diabetic nephropathy has shown satisfactory results rat models by the increase of fasting insulin levels. The serum levels of creatinine and blood urea nitrogen were also reduced after EX-4 administration. It seems that Ex-4 has the ability to alleviate oxidative stress via decreasing malondialdehyde production and decrease of lipid peroxidation rate while the bioactivity of superoxide dismutase and glutathione peroxidase were also induced. One of the most therapeutic aspects related to Ex-4 therapeutic effects correlates with the anti-inflammatory property via the control of TNF-α, IL-6, hypersensitive C-reactive protein and CCL5 as commonly described in end-stage renal failure. Scientific literature highlighted the potent role of Ex-4 in alleviating renal proximal tubular cell injury in diabetic mice. It was established that the levels of IL-1β, TNF-α and ROS content and malondialdehyde (MDA) levels were increased in renal tissue with the progression of disease. The development of diabetic condition predisposes the initiation of tubulointerstitial fibrosis that hastens renal failure. During T2DM hyperglycemic condition, the accumulation of extracellular matrix is extensively promoted in spaces between renal tubular epithelial cells. The occupation of connective tissue with the extracellular matrix can make multiple resident renal cells to release extracellular vesicles containing proteins, mRNA, and miRNA, contributing to the progression of fibrotic changes. The application of Ex-4 decreases diabetic-associated fibrosis by decreasing TGF-β. Along with these changes, the proliferation of fibroblasts and collagen deposition are decreased in renal niche. Another modulatory effect of Ex-4 in reducing diabetes associated fibrosis could be related to changes in extracellular vesicle cargoes. It seems that the type and intensity of specific miRNAs and factors are modulated that subsequently decreases diabetic-fibrosis.Diabetic nephropathy is the consequent of T2DM. The elevation of excessive glucose contents injures the renal filtration system, thereby contributing to the occurrence of proteinuria or macroalbuminuria.vice versa. CRS is classified into five subtypes  and oxidative stress  markers. Besides, the synthesis of apoptosis , fibrosis (Samd3 and TGF-β) markers and DNA damage (γ-H2AX) is inhibited after Ex-4 and melatonin treatment. Pathological features were decreased after using Ex-4 and melatonin in CRS patients which indicated the effective impact of this method on kidney function.In addition to diabetes-related nephropathies, cardio-renal syndrome (CRS) is defined as a common disorder between heart and kidneys in which any abnormalities in one of these organs may impact other tissue and subtypes .89 As Furthermore, Ex-4 along with melatonin improved mitochondrial function and suppressed βMHC mRNA/protein expression, resulting in the alleviation of cardiac hypertrophy, is decreased. These findings indicated the compensatory role of Ex-4 and melatonin treatment on cardiac function. A close relation between T2DM and NAFLD has been previously well-established in which insulin-resistance and compensatory hyperinsulinemia are commonly seen by defective lipid metabolism and accumulation of triglyceride in hepatic tissue in patients with NAFLD or T2DM associate β-cell dysfunction.Due to the high-rate metabolic activity of hepatic tissue, this tissue is eligible for different metabolic disorders and diseases. Non-alcoholic fatty liver diseases (NAFLDs) are clinically determined with the accumulation of byproduct three acyl glycerol (TAG) in the hepatic tissue that coincided with the development of intracytoplasmic lipid droplets affecting over 5% of total hepatocytes. Beside, an excessive lipid accumulation causes to hepatic cells lipotoxicity and oxidative stress via increased lipid peroxidation, mitochondrial insufficiency, and high content of ROS production. In patients with T2DM, both glucose and lipid metabolisms are deteriorated, leading to the increase of plasma levels of low-density lipoprotein, and adversely reduction of high-density lipoprotein. In the case of NAFLD, the content of different serum transaminases is slightly raised with NAFLD and the administration of GLP-1R could decrease systemic transaminase levels via the promotion of hepatic lipid metabolism. One possible mechanism is that hepatic lipid metabolism is activated via triggering GLP-1R signaling cascade and the promotion of PPAR-α. These changes diminish the synthesis of apolipoprotein C which accelerates the degradation of fatty acid in plasma, after the use of Ex-4 and stimulation of GLP-1R signaling pathway. Experiments have shown that incubation of ob/ob mice with Ex-4 exerted anti-hepatic steatosis and -fibrosis capacities. The modulatory effect of Ex-4 on hepatic steatosis is done by reducing lipid droplets. The deposition of collagens along the hepatic sinusoids in the fibrotic liver is reduced after Ex-4 treatment. It was found that Ex-4 increased the reduced PPAR-α and glucose transporter 4 expressions in ob/ob mice. The activity of FTO correlates with energy homeostasis and the control of energy consumption. Tha is gene is commonly upregulated in NFLD candidates, contributing to oxidative stress indicated by an increased MDA production and decrease of superoxide dismutase activity. Meanwhile, the rate of lipogenesis is increased inside hepatocytes. Under these conditions, the basal dynamic of effectors such as PI3K and Akt is prohibited.Along with these changes, NAFLD can lead to hepatic steatosis, NASH, hepatic cirrhosis, and hepatocellular carcinoma.FTO expression and MDA production and restoration Superoxide dismutase activity. In addition, the expression of genes participating in fatty acid oxidation promotes hepatocyte functional behavior after phosphorylation of Akt. As above-mentioned, steatosis is routinely reported in the NFLD patients correlated with an increased lipogenesis rate and TAGs accumulation inside hepatocytes.In NFLD animal models, the use of Ex-4 leads to alleviation of NFLD related complications via suppression of  in vitro model of steatosis. Ex-4 has the ability to activate β-catenin which in turn stops lipogenesis by suppression of PPARγ and SREBP-1. The use of Ex-4 in steatoric patients remove excessive fatty acids via regulating lipid metabolism and reducing VLDL synthesis after suppression of genes such as peroxisome Pgc1β, transcription factor Srebp-1c and Fasn, Dgat1, and Apob. Fasn participates in de novo lipogenesis and Dgat1 promotes the last steps in hepatic three glyceride synthesis.It is thought that the promotion of Wnt/β-catenin signaling could prohibit adipogenesis by suppressing CCAAT/enhancer-binding proteins-induced PPARγ expression in which a decrease in the expression of β-catenin was unveiled inin vivo and in vitro modelscoincided with nicotinamide phosphoribosyl transferase (Nampt) activity. The Nampt/visfatin enzyme could catalyze the conversion of NAM toward NAD+ and is required for Sirt-1 activity.It was found that Sirt-1 expression is decreased in high-fat-diet animal models and Ex-4 could reverse the Sirt-1 function either in  Disruption of ER homeostasis contributes to the accumulation of misfolded/unfolded proteins seen in obesity. Dysfunction of insulin function and T2DM also has a pivotal role in fatty liver disease development. Progressive ER stress contributes to the development of NFLD and nonalcoholic steatohepatitis by different molecular mechanisms, including lipogenesis, inflammation, apoptosis, and autophagy. Documents showed that Ex-4 can alleviate hepatic steatosis and ER stress via controlling lipin-1β/α ratio and lipid hemostasis through Sirt1 and AMPK signaling pathways. Hepatic glycoproteins like SEPP1 and fetuin-A are known as novel biomarkers who participated in insulin resistance and NFLD. Overactivity of these glycoproteins in palmitic acid-treated hepatocytes contributes to ER stress after Ex-4 treatment via activating Sirt-1 and AMPK expression.As previously mentioned, endoplasmic reticulum (ER) plays a critical role in lipid and protein biosynthesis.According to versatile functions of Ex-4 in different tissues and similar activities to GLP-1, it seems that the use of Ex-4 is possibly helpful in the modulation of pathologies correlated with insulin dysfunction and the promotion of GLP-1R signaling pathway.Not applicableThe authors have no conflict of interest to declare.


CX3CR1 is a G protein-coupled receptor that is expressed exclusively by microglia within the brain parenchyma. The only known physiological CX3CR1 ligand is the chemokine fractalkine (FKN), which is constitutively expressed in neuronal cell membranes and tonically released by them. Through its key role in microglia-neuron communication, the FKN/CX3CR1 axis regulates microglial state, neuronal survival, synaptic plasticity, and a variety of synaptic functions, as well as neuronal excitability via cytokine release modulation, chemotaxis, and phagocytosis. Thus, the absence of CX3CR1 or any failure in the FKN/CX3CR1 axis has been linked to alterations in different brain functions, including changes in synaptic and network plasticity in structures such as the hippocampus, cortex, brainstem, and spinal cord. Since synaptic plasticity is a basic phenomenon in neural circuit integration and adjustment, here, we will review its modulation by the FKN/CX3CR1 axis in diverse brain circuits and its impact on brain function and adaptation in health and disease. CX3C chemokine receptor 1 (CX3CR1) is a seven-transmembrane domain G protein-coupled receptor involved in neuron-microglia crosstalk within the central nervous system (CNS)  and leukThere are two isoforms of FKN, one is soluble and the other is anchored to the membrane , 21  overproduction [β-induced neuronal damage and amnesia in AD animal models [β levels [β bidirectionally interact. For instance, CX3CR1 deficiency induces a reduction in Aβ accumulation in AD transgenic mice [β administration leads to an increase in CX3CR1 levels [β-induced microglial activation, synaptic plasticity blockade, and memory impairment are also reduced [Alterations in the FKN/CX3CR1 axis play a role in Alzheimer's Disease (AD), a neurodegenerative disorder closely associated with aging, amyloid beta  [While the levels of CX3CR1 exhibit complex, even opposite, changes in Parkinson Disease (PD) , 121, thgression . The FKNgression , which agra (SN)  and are gra (SN)  and a lagra (SN)  as well gra (SN) . FKN cangra (SN)  or MPTP gra (SN) , or whengra (SN) . In agregra (SN) , when algra (SN)  or in PDgra (SN) . Howevergra (SN) . Moreovegra (SN) . Thus, igra (SN) , 124. Thgra (SN) .Q111 immortalized embryonic striatal cell line [The FKN expression is reduced in the putamen of patients with Huntington's Disease (HD)  and in Rell line  and the ell line . Moreoveell line . Reducedell line . Interes−/− mice compared to wild-type animals [FKN and CX3CR1 levels are increased in epilepsy patients and rodent epilepsy models , 131–134 animals . Researc animals . Moreove animals . Thus, i animals , 131. Ep animals , 102–105κB, and inflammasome activation, which leads to the lessening of infarct size and the attenuation of neurological deficits induced by the MCAO model [FKN levels are reduced after stroke in patients  and afteAO model , 141, whAO model . HoweverAO model . MoreoveAO model , clearlyAO model .These findings coincide with the reduced inflammation, brain injury, and mortality found in other reports after the induction of MCAO in FKN- or CX3CR1-deficient mice , 146, whThe FKN/CX3CR1 axis also plays a part in the regulation of emotional behavior and mood disorders. While a lower CX3CR1 expression has been found in patients with bipolar disorder , changesβ signaling [From the variable changes in the FKN/CX3CR axis observed under the pathological conditions just reviewed, which seem to be mediated by TGF-ignaling , it is fβ, TNF-α, NO, and IL-6, which were attenuated by FKN treatment [α, IL-1β, and cC1q [−/− mice compared to wild-type animals [β expression compared to wild-type mice [−/− mice compared to wild-type animals [The FKN/CX3CR1 axis is a strong modulator for the production and release of proinflammatory microglial cytokine synthesis and the microglial response to immunogens , 36, 159reatment , 36, 159and cC1q , 52, 160and cC1q , 161–163ype mice , 164. Be animals , 160, su animals , 160.β and TFN-α [β, IL-6, and TNF-α [The reduction of CX3CR1 signaling can also impact neural function under conditions of mild inflammation , such asnd TFN-α . In thisnd TFN-α , 165. Mond TNF-α   in proportion to miniature synaptic currents is also blocked in CX3CR1−/− mice [−/− mice, CA3–CA1 functional connectivity remains diminished due to a small number of functional synapses between Schaffer collaterals and CA1 pyramidal neurons or to a decrease in their synaptic strength [−/− mice that dentate gyrus–CA3 connections remain diminished [−/− mice [The exact signals between microglia and neurons used to regulate synaptic activity are not completely understood but clearly include the FKN/CX3CR1 axis  Figures. Since t−/− mice , which p−/− mice . Consequstrength , 45. We minished , which iminished , negativ−/− mice .−/− mice exhibit a reduction in the amplitude of sEPSC recorded in CA3 pyramidal neurons along with a reduction in the amplitude of mossy-fiber evoked EPSC [Acute FKN induces a reduction in synaptic transmission reflected as the amplitude of field excitatory postsynaptic potentials (fEPSPs), in a concentration-dependent manner , 198, anked EPSC . Howeverked EPSC  or by thked EPSC , 197. Adked EPSC , 79, 198ked EPSC . Moreoveked EPSC . This efked EPSC .Many studies have focused on the influence of microglia on long-term potentiation (LTP) and long-term depression (LTD) , 200–202−/− animals [β by administering the antagonist of its receptor (IL-1RA) is sufficient to reestablish this long-term synaptic plasticity [−/− animals have enhanced LTP when induced by a weak stimulation protocol [FKN administration also inhibits hippocampal LTP  when app animals , 45, 153 animals , 45. Intasticity  . For inβ, and subsequently promotes pain [β through the antagonist of its receptor IL-1RA reduces chronic pain, suggesting that IL-1β accumulation triggered by microglial activation induces pain facilitation [β. They described that the injury signal activates CX3CR1 and subsequently p38 MAPK kinase, culminating in the release of IL-1β that worsens pain in mice. Similarly, the induction of spinal facilitation by FKN is independent of TNF-α but dependent on IL-1β, since nociceptive facilitation is blocked by antagonizing the action of IL-1β prior to the addition of FKN [CX3CR1 activation leads to the activation of the p38 MAP kinase pathway, which is critical for pain development  Figure  as it intes pain , 213 (Fites pain . Particulitation , 215. Win of FKN  (Figure Reducing CX3CR1 activity by either antagonizing the receptor  or using−/− animals [β, IL-6, and TNF-α after SCI and facilitating recovery from spinal cord damage and decreasing pain, we can conclude that the FKN/CX3CR1 axis could be considered a therapeutic target to relieve chronic pain (Regarding FKN/CX3CR1 axis involvement in spinal plasticity, the induction sciatic nerve LTP is inhibited with the use of anti-CX3CR1 antibodies in wild-type animals and is not observed in CX3CR1 animals . Additio animals . Based o animals  showing nic pain .in vitro [A vital function that is highly sensitive to inflammation and microglial modulation is breathing , 217, whin vitro . Similarin vitro . As alrein vitro .β [in vivo [in vitro [We have also found that activating CX3CR1 with FKN induces a depression of the respiratory rhythm generated by the preBötC , which iβ  and whic[in vivo . Moreove[in vivo , 226. Fo[in vivo . Finallyin vitro , which iin vitro , indicatIn this review, we summarized information on how the CX3CR1 receptor modulates microglial activity and how this modulation is affected by a variety of factors, including the brain structure –45, anim


Aberrant activation or suppression of WNT/β-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and “dark.” Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the β-catenin–dependent and β-catenin–independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated β-catenin–dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor–related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase–dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor–related protein 6 phosphorylation and β-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway. WNT signaling regulates embryonic development, injury repair, regeneration, and tissue homeostasis , 2. WNT Kinases, a highly druggable class of proteins, are master regulators of various signaling pathways, including WNT signaling. WNT/β-catenin-dependent signaling is often described with respect to the main effector protein, β-catenin. In the absence of WNT ligand, a destruction complex comprised of casein kinase 1α , glycogen synthase kinase 3β (GSK3β), AXIN1/2, and APC phosphorylates β-catenin resulting in its subsequent ubiquitylation and degradation by β transducin repeat–containing protein (β-TRCP) and the proteasome , 7, 8. Ihttps://commonfund.nih.gov/idg/index) (A). Studies suggest that the palmitoylation modification positions CK1γ kinases to act within the WNT signalosome, as other CK1 family members are not anchored to the membrane  functions within β-catenin-independent signaling to regulate Van Gogh phosphorylation at the cell membrane, as well as regulating Notch pathway activity (The CK1γ subfamily of kinases is comprised of three genes: CK1γ1 (CSNK1G1), CK1γ2 (CSNK1G2), and CK1γ3 (CSNK1G3). All three are identified as understudied and dark kinases by the National Institutes of Health initiative, Illuminating the Druggable Genome (g/index) . The CK1g/index) A. Studiemembrane . Apart fmembrane . In addimembrane . Within membrane . Interesmembrane . In Drosactivity , 27, 28.Following WNT ligand–induced FZD–LRP6 co-complex, disheveled (DVL) is recruited to the intracellular tail of FZD . This biHere, we defined protein–protein proximity networks for the CK1γ family and functionally evaluated their contribution to a small panel of disparate signaling pathways. Each CK1γ family member co-complexed with various WNT components, including β-catenin and core members of the planar cell polarity complex. Surprisingly, only CK1γ3 activated β-catenin-dependent WNT signaling when overexpressed. Our experiments reveal a family-specific role for CK1γ3 in promoting phosphorylation of LRP6 within the WNT signalosome. In addition, we characterized two pan-CK1γ–specific inhibitors. Genetic or chemical inhibition of the CK1γ family suppresses WNT signaling, but suppression of CK1γ3 alone had no effect. This finding was extended and confirmed with a kinase-dead CK1γ3 mutant, which suppressed WNT signaling.B). Streptavidin affinity purification followed by W. blot analysis confirmed proximal complex formation between CK1γ family members and WNT pathway proteins LRP6, AXIN1, and β-catenin (CTNNB1) . Next, in biological triplicate, we treated the stable cell lines with biotin for 30 min before streptavidin purification and MS analysis. Resulting identifications were probabilistically scored against controls with Significance Analysis of INTeractome (SAINT) and then further ranked with CompPASS before visualization (B and C) . Of 548 high-confidence protein interactions for CK1γ1, CK1γ2, and CK1γ3, 326 proteins were seen with all subfamily members. β-catenin, ZDHHC8 , and PIK3R3 (phosphoinositide-3-kinase regulatory subunit 3) were the most abundant proximal proteins across all three networks. Several core components of the β-catenin-dependent and β-catenin-independent signaling pathway were also identified, such as DVL, APC, cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), and VANGL planar cell polarity protein 1 . To further strengthen and expand the proximity networks, we repeated the experiment at 120 min of biotin treatment, revealing LRP6 and FZD3 as CK1γ3-proximal proteins . To further explore these networks and their connectivity to the WNT pathway, we annotated all proteins for functional contribution to β-catenin-dependent transcription. From four recently published independent genetic screens, 1143 genes were identified as functionally impactful to β-catenin-dependent transcription . Last, we visualized the data as a network to examine high-confidence prey–prey interactions and subcellular localization . The majority of the prey proteins are known to localize to the plasma membrane.To better understand each member of the CK1γ family, we defined their protein–protein proximity networks and functionally evaluated their impact on signal transduction. We used the miniTurbo (mT) promiscuous biotin ligase to comprehensively map CK1γ proximal proteins, which requires a shorter biotin labeling window compared with BirA∗ , 35. SpeFig. 1B and C) , 37, 38.cription  39, 40,, 40,B. Scription , 42. IntF). CK1γ2 and CK1γ3 interacted with β-catenin-dependent WNT signaling components , whereas CK1γ1 interacted with these proteins but to a lesser extent. WNT3A stimulation increased pLRP6 S1490 in all three CK1γ pulldowns. Additional WNT3A-dependent changes were not observed. We also confirmed the interaction between the CK1γ family and proteins involved in β-catenin-independent signaling, CELSR2 and prickle planar cell polarity protein 1 (F).To confirm the MS results, and to test the impact of WNT3A stimulation, we analyzed the CK1γ affinity-purified proximal proteins by W. blot analysis F. CK1γ2 rotein 1 F.A) (B). In the presence of WNT3A conditioned media (CM), CK1γ1 and CK1γ2 activated WNT signaling similar to control, whereas CK1γ3 overexpression increased activation of BAR (C) (D) , Notch, transforming growth factor beta (SMAD), NRF2 , retinoic acid (retinoic acid receptor), and tumor necrosis factor alpha (NF-κB) signaling pathways A 15, 43, 43A (15A) , 45. SurA) B. In then of BAR C 19, 20, 20A (B) BAR (C) D  (F). HEK293T cells lacking DVL1, DVL2, and DVL3 did not respond to WNT3A CM, as expected (F). These data indicate that WNT3A ligand secretion and expression of LRP5/6 are required for CK1γ3-driven activation of β-catenin-dependent transcription; the DVL proteins are involved but are not absolutely required.To evaluate which WNT pathway proteins are required for CK1γ3 activation of β-catenin-dependent transcription, we studied established inhibitors of WNT signaling and KO cell lines. While overexpression of CK1γ3 activated BAR in the absence of WNT3A CM, treatment with C59, a PORCN inhibitor , blocked this activation, which supports a role for autocrine WNT3A signaling in HEK293T cells E 46). W. WE) (46E) F. HEK293expected . Surprisexpected F. These A). CK1γ1 overexpression did not increase LRP6 phosphorylation at T1479 or the GSK3β site, S1490, irrespective of WNT3A treatment (B and C). With CK1γ2 overexpression, S1490 LRP6 phosphorylation increased in response to WNT3A but not T1479. CK1γ3 overexpression robustly increased LRP6 phosphorylation at T1479, as well as S1490, in both the absence and presence of WNT3A. Together, these data suggest that in contrast to CK1γ3, CK1γ1 and CK1γ2 do not activate WNT signaling in the absence of exogenous WNT3A ligand and do not induce LRP6 phosphorylation at T1479.It was previously reported that overexpression of CK1γ1 together with overexpression of LRP6 and DVL resulted in LRP6 phosphorylation at T1493 and T1479 , 20, 48.Fig. 3A). In the presence of WNT3A CM, kinase-dead CK1γ3-K72A expression suppressed BAR activity as compared with WT CK1γ3 or control, which is supportive of dominant-negative activity (A). In an orthogonal experiment, CK1γ3 or CK1γ3-K72A was expressed in HEK293T cells stably harboring a BAR-GFP fluorescent reporter. Live cell imaging over 3 days revealed that CK1γ3-K72A suppressed WNT3A-driven β-catenin-dependent transcription (B). Last, we tested whether expression of the CK1γ3 kinase-dead mutant impacted the phosphorylation of LRP6. CK1γ3 overexpression resulted in LRP6 phosphorylation at both S1490 and T1479 sites following WNT3A CM exposure (C–E). In contrast, overexpression of CK1γ3-K72A suppressed phosphorylation of LRP6 at both sites. These data demonstrate that kinase activity of CK1γ3 is required for phosphorylation of LRP6 and activation of the WNT pathway.We next performed loss-of-function studies to determine if the CK1γ family was required for WNT3A-driven β-catenin-dependent transcription. First, we generated a CK1γ3 kinase-dead mutant (CK1γ3-K72A). In contrast to CK1γ3-WT, expression of kinase-dead CK1γ3-K72A in HEK293T cells did not induce the activity of the BAR transcriptional reporter A. In theactivity A. In an cription B. Last, exposure , C–E. InA). Transfection of the indicated siRNA into HEK293T cells stably harboring BAR revealed that knockdown of CK1γ3 alone or a pool of CK1γ1/2 did not significantly inhibit WNT activation. However, knockdown of CK1γ1/2/3 suppressed WNT activation by approximately 50% (B). Second, we examined β-catenin protein stabilization and LRP6 phosphorylation following a WNT3A time course. β-catenin protein levels increased in the control cells. CK1γ3 knockdown modestly suppressed WNT3A CM-induced β-catenin stabilization. With simultaneous knockdown of CK1γ1/2/3, stabilization of β-catenin was significantly impaired as compared with control siRNAs (C and D). Finally, knockdown of CK1γ3 and CK1γ1/2/3 decreased phosphorylation of LRP6 at T1479 and S1490 compared with the control knockdown .To extend this finding, we tested the effect of CK1γ knockdown on BAR reporter activity. First, we confirmed knockdown by quantitative PCR of two nonoverlapping siRNAs for CK1γ3 as well as pooled siRNAs targeting CK1γ1/2/3 or CK1γ1/2 A. Transftely 50% B. Secondl siRNAs , C and DA and B) (referred to as AKI throughout) for kinome-wide profiling against 403 WT kinases at DiscoverX at 1 μM. This compound demonstrated modest selectivity, with a calculated S10(1 μM) = 0.042, corresponding to 17 kinases with a Percent of Control (PoC) <10 at 1 μM (C). GSK3α and GSK3β were among those kinases potently inhibited as well as all three CK1γ kinases (D). To confirm this, we collected the corresponding enzymatic assay activity for all three CK1γ family members and found AKI to inhibit all three enzymes with IC50 values ≤275 nM (B). Finally, a cellular target engagement assay (NanoBRET) was used to measure the potency of enzymatic inhibition for CK1γ2 in live cells. CK1γ1 and CK1γ3 NanoBRET tools are not yet available. We observed modest suppression of CK1γ2 in the cell-based system: IC50 = 991 nM (E).To complement the genetic study of CK1γ family within the WNT pathway, we sought CK1γ chemical inhibitors. Molecular tool compounds that target the larger CK1 family of kinases have been studied within WNT signaling in the past . However at 1 μM C. GSK3α  kinases D. To con ≤275 nM B. Finall= 991 nM E.Figure A) (referred to as FP1 throughout) . While Ambit KINOMEscan data at 1 μM were included as supporting information in the original publication, kinome-wide selectivity data were lacking (10(1 μM) = 0.035 . Among the potently inhibited kinases are all three CK1γ family members, CK1δ, CK1ε, and CK1α . NanoBRET was used to test the enzymatic  and binding  assay activities for the CK1γ2 kinase in cells .Because the AKI compound also targeted GSK3β, we turned to the literature for an alternative chemotype with published potency against the CK1γ kinase subfamily . Amgen rFig. 7oughout) . We firs lacking . We sent = 0.035 , correspin cells . Potency50 = 7.14 nM (A). AKI was significantly more potent on GSK3 kinases than on CK1γ2. In contrast, FP1 was inactive up to 10 μM in the GSK3β NanoBRET assay (B). As a control, APY69, validated as active in the GSK3 NanoBRET assay by Promega, was included in both the CK1γ2 and GSK3β NanoBRET assays. APY69 had an IC50 = 0.23 nM in the GSK3β NanoBRET assay and was inactive in the CK1γ2 NanoBRET assay. To confirm that AKI inhibits GSK3β, we compared it in a BAR assay with the GSK3β inhibitor, CHIR99021 (C) . B. B50 = 7D). In agreement with this, treatment of cells with FP1 and WNT3A CM resulted in diminished β-catenin stabilization and phosphorylation of LRP6 at site S1490, as compared with the dimethyl sulfoxide control-treated cells . Importantly, when cells were treated with FP1 and then exposed to WNT3A, LRP6 phosphorylation at T1479 did not increase (E and H).Finally, we tested whether FP1 impacted CK1γ3 activation of WNT signaling in a BAR assay. We overexpressed CK1γ3 and treated with increasing doses of FP1 and observed a decrease in BAR activity D. In agrincrease , E and HProtein kinases govern information flow though cellular signaling networks and ultimately impact all cell biology. Their exceptional druggability and centrality within human disease networks has supported decades of research and therapeutic development . HoweverDrosophila, the ZDHHC8 ortholog palmitoylates Scribble, which is a key component of the planar cell polarity pathway and has been shown to negatively regulate WNT/β-catenin signaling in human cell lines . Also similar to our results, without co-overexpression, the authors observed minimal activation of WNT signaling with CK1γ1 overexpression (E). Together, these data build upon prior work to support a role for the CK1γ family and specifically CK1γ3 as important regulators of WNT signaling.The observed physical and functional connections to β-catenin-dependent and β-catenin-independent WNT signal transduction are unsurprising. Previous studies demonstrated that CK1γ1 activated β-catenin-dependent WNT signaling with co-overexpression of LRP6 or DVL , 20. Imppression . Subsequpression E. TogethLoss-of-function studies for the CK1γ family within the WNT pathway have not been reported, perhaps because of modest effect sizes. Functional redundancy within the WNT signaling pathway is well acknowledged, and indeed our data support redundancy for the CK1γ family , 64, 65.Beyond genetics, we evaluated two tool chemical inhibitors for the CK1 family. The AKI compound also targeted GSK3β, which complicates the study of CK1γ in WNT signaling . The othA). Five β-sheets on the C terminus of the CK1γ subfamily proteins create an open crescent moon shape : HEK293T/17 , RKO , Lcells , and WNT3A-expressing Lcells . All cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Each cell line was tested for mycoplasma contamination and passaged no more than 20 passages from the original ATCC stock.All cells were cultured at 37 °C and 5% COCM was collected as described by ATCC. Briefly, WNT3A and control Lcells were grown to 100% confluency before fresh media were added conditioned for 48 h and then collected.Radioimmunoprecipitation assay (RIPA) lysis buffer contains 10% glycerol, 50 mM Tris–HCl, pH 7.4, 150 mM sodium chloride (NaCl), 2 mM EDTA, 0.1% SDS, 1% Nonidet P-40, and 0.2% sodium deoxycholate. TAP lysis buffer contains 10% glycerol, 50 mM Hepes (pH 8), 150 mM NaCl, 2 mM EDTA, and 0.1% Nonidet P-40. Triton lysis buffer contains 10% glycerol, 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, and 1% Triton X-1000.mT constructs (UBC-mT-V5-CONSTRUCT) were generated by LR multisite recombination  utilizing the PEL system . CK1γ3 kCK1γ3 gib A—Forward: 5′-CTT TTT TAT AAT GCC AAC TTT GTA CAA AAA AGT TGG CAT GGA GAA CAA GAA GAA GGA CAA GGCK1γ3 K72A gib A—Reverse: 5′-GCTCCAGCGCGATGGCCACCK1γ3 K72A gib B—Forward: 5′-GTGGCCATCGCGCTGGAGCCK1γ3 gib B—Reverse: 5′-TTT CTT ATA ATG CCA ACT TTG TAC AAG AAA GTT GGA CTA CTT GTG TCT CTG GAT TGT CTT CCStandard W. blot techniques were utilized and performed as previously described , 43. BriRenilla normalized values were averaged across triplicates to yield the data presented and standard error. Each assay was repeated in biological triplicate, unless otherwise stated. Firefly luciferase and the Renilla control were detected using the Promega Dual-Luciferase Reporter Assay System per the manufacturer’s protocol . Plates were read on the Victor Nivo plate reader (PerkinElmer).All luciferase reporter assays were performed as previously described , 43. BriAll siRNAs were obtained from Life Technologies (Thermo Fisher Scientific). Cell lines were used and transfected with RNAiMAX  and for 72 h before W. blot analysis. Sequences are listed in Total RNA was extracted from RKO and HT1080 cells using Invitrogen PureLink RNA mini kit  following the manufacturer’s manual. The AXIN2 and NKD1 primers are described . All othCells were incubated for indicated time with 50 mM biotin, washed 3× in cold PBS, then lysed in RIPA lysis buffer, flash frozen on dry ice, then thawed, and incubated on ice for 30 min. Lysates were then sonicated for 20 s in 5 s pulses. Samples were then high speed cleared, and protein concentration was determined by bicinchoninic acid. About 50 μl of bead slurry per sample was washed 5× in RIPA lysis buffer and then incubated with lysate at 4 °C with rotation for either 1 h for W. blot analysis or overnight for MS analysis. Beads were then washed 5× with RIPA lysis buffer. For W. blot analysis, samples were then eluted in a 1:1:1 mixture of 1 M DTT, 4× SDS loading buffer, and RIPA lysis buffer and heated at 70 °C for 10 min.g for 2 min at RT to remove the supernatant containing protein from the streptavidin beads. The supernatant was transferred to a new tube, and 2.5 μg of MS-grade trypsin was added to each sample. The samples were incubated at 37 °C overnight, and then HCl was added to the sample at a final concentration of 250 mM and incubated for 45 min at 37 °C to deactivate the trypsin. Pierce C18 spin columns  were placed in a 2 ml centrifuge tube, the column was activated by adding 200 μl of 100% acetonitrile (ACN) and centrifuged at 3000g for 2 min. Columns were equilibrated by adding 200 μl of 0.5% TFA and centrifuged at 3000g for 2 min. This step was then repeated. The sample was resuspended in 200 μl of 0.5% TFA, then loaded onto the column, and centrifuged for 5 min at 1000g. The sample was reloaded onto the column and centrifuged a second time for 5 min at 1000g. The column was washed twice with 200 μl of 5% ACN/0.5% TFA and centrifuged for 2 min at 3000g. The samples were eluted by adding 50 μl 70% ACN and centrifuged for 5 min at 1000g. A second elution step was performed by adding 50 μl 70% ACN and centrifuged for 2 min at 3000g. Following a C18 clean up, an ethyl acetate clean up was performed. The sample was resuspended in 100 μl of 0.1% TFA, then 1 ml of water saturated ethyl acetate was added to each sample, vortexed, and centrifuged at maximum speed for 5 min. The upper layer of ethyl acetate was removed. This process was repeated for a total of three times. Samples were then dried down in the speed vac and resuspended in 25 μl of 98/2 buffer A (water + 0.1% formic acid [FA])/buffer B (ACN + 0.1% FA).Following the affinity pulldown (described previously), beads were washed 2× with RIPA, 2× with TAP lysis buffer, and 3× with 50 mM ammonium biocarbonate buffer. About 1 mg of RapiGest SF surfactant  was resuspended in 200 μl of 50 mM to give a 0.5% RapiGest solution. Streptavidin beads were resuspended in 100 μl of 0.5% RapiGest and vortexed. About 1 M DTT in ammonium biocarbonate was added to the sample for a final concentration of 5 mM, and samples were heated at 60 °C for 30 min. After allowing samples to cool to room temperature, chloroacetamide was added to each sample for a final concentration of 15 mM and incubated in the dark for 20 min at RT. The samples were then centrifuged at 400via reverse-phase nano-HPLC using an RSLCnano Ultimate 3000 (Thermo Fisher Scientific). The mobile phase consisted of water + 0.1% FA as buffer A and ACN + 0.1% FA as buffer B. Peptides were loaded onto a μPAC Trapping column (PharmaFluidics) and separated on a 200 cm μPAC column (PharmaFluidics) operated at 30 °C using a 100 min gradient from 2% to 25% buffer B, followed by a 20 min gradient from 25% to 35% buffer B, flowing at 300 nl/min. MS analysis was performed on an Orbitrap Eclipse (Thermo Fisher Scientific) operated in data-dependent acquisition mode. MS1 scans were acquired in the Orbitrap at 120 k resolution, with a 250% normalized automated gain control target, auto maximum injection time, and an m/z scan range of 375 to 1500. Both the linear ion trap and the Orbitrap were used for MS2 scans. MS2 targets with a charge of +2 or +3 and >90% precursor fit at either an m/z 0.8 or m/z 0.4 -wide isolation window were fragmented with by collision-induced dissociation at 35% collision energy and scanned in the linear ion trap at the widest window width that passed the thresholds. All remaining MS2 targets with charge from +2 to +6, >5e4 intensity, and >10% precursor fit at isolation widths of m/z 1.6 , m/z 0.8 , or m/z 0.4 wide were fragmented with higher-energy collision dissociation at 30% collision energy and scanned in the orbitrap at 15 k resolution at the widest window width that passed the thresholds. MS2 automated gain control targets and maximum injection times were set to standard and auto for their respective analyzers. Dynamic exclusion was set to 60 s. Acquisition was performed with a 2.7 s cycle time. For 120 min biotin-treated samples, the same method was used except the following: peptides were separated on a 50 cm μPAC column using a 115 min gradient from 2% to 25% B followed by a 5 min gradient from 25 to 30% B, MS1 scans were acquired at 240 k resolution, and an m/z 375 to 2000 scan range. All MS2 scans were acquired in the linear ion trap using an m/z 0.7-wide isolation window and fragmented with higher-energy collision dissociation at 35% collision energy. Acquisition was performed with a 1.0 s cycle time.Trypsinized peptides from 30 min biotin-treated cells were separated https://github.com/dnusinow/cRomppass), validated WNT-related proteins from the literature, and hits from published siRNA and CRISPR screens for WNT pathway regulation   using thgulation , 41, 42.gulation : ≤0.01 Fr et al. , 41, 42.Nov 2021 .FP1-24-2 was prepared as previously described . Detailsin vacuo and purified by preparative HPLC (10–100% MeOH in H2O (+0.05% TFA) to yield the desired product 2-methyl)-5-methoxypyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolopyridin-4-one as an off-white solid .To a flask was added 2-bromo-1-methyl)-5-methoxypyridin-4-yl)ethan-1-one , piperidine-2,4-dione , and ammonium acetate  in ethanol , and the reaction was stirred at room temperature for 16 h. The reaction was concentrated 1H NMR : δ 8.30 , 7.77 , 7.22 to 7.13 , 6.99 , 6.85 to 6.80 , 5.08 , 4.09 , 3.57 , 2.97 . 13C NMR : δ 169.0, 152.1, 150.1, 140.8, 134.4, 130.3, 127.7, 119.3, 118.48, 118.46, 118.29, 118.28, 115.5, 111.74, 111.70, 111.68, 111.64, 110.5, 105.7, 105.5, 72.0, 57.0, 41.7, 23.0. 19F NMR : δ −138.23 – −138.37 (m), −150.69 . LC–MS: expected mass for [M + H]+ (C20H18F2N3O3), m/z 386.12; found, m/z 386. LC–MS and 1H NMR: >95%.Km = ATP in dose–response (9-pt curve in duplicate) for each CK1γ kinase listed in Figures 6B and 7B. Details related to the substrate used, protein constructs, controls, and assay protocol for each kinase assay are included on the Eurofins website: https://www.eurofinsdiscoveryservices.com/.Eurofins' kinase enzymatic radiometric assays were carried out at the scanMAX assay platform was used to assess the selectivity of FP1-24-2 and AKI00000062a at 1 μM at Eurofins DiscoverX Corporation. As described previously, this commercial assay platform screens against 403 WT human kinases in binding assays and provides PoC values  and GSK3β (NLuc-GSK3β) were kindly provided by Promega. NanoBRET assays were carried out as described previously  were purchased from ATCC. These cells were grown in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (Corning). They were incubated in 5% COeviously . Preferrvia the PRIDE partner repository with the dataset identifier PXD032138 (The MS proteomics data have been deposited to the ProteomeXchange Consortium XD032138 .This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Burkholderia (B.) mallei, have not been assessed in detail and are complicated by the high clonality of this organism. Among the identification and typing methods, PCR-based methods for distinguishing B. mallei from its close relative B. pseudomallei as well as genotyping using tandem repeat regions (MLVA) are established. The advent and continuous advancement of the sequencing techniques and the reconstruction of closed genomes enable the development of genome guided epidemiological tools. For achieving a higher genomic resolution, genotyping methods based on whole genome sequencing data can be employed, like genome-wide single nucleotide polymorphisms. One of the limitations in obtaining complete genomic sequences for further molecular characterization of B. mallei is its high GC content. In this review, we aim to provide an overview of the widely used detection and typing methods for B. mallei and illustrate gaps that still require development. The genomic features of Burkholderia, their high homology and clonality will be first described from a comparative genomics perspective. Then, the commonly used molecular detection (PCR systems) and typing systems  will be presented and put in perspective with recently developed genomic methods. Also, the increasing availability of B. mallei genomic sequences and evolution of the sequencing methods offers exciting prospects for further refinement of B. mallei typing, that could overcome the difficulties presently encountered with this particular bacterium.Although glanders has been eradicated in most of the developed world, the disease still persists in various countries such as Brazil, India, Pakistan, Bangladesh, Nepal, Iran, Bahrain, UAE and Turkey. It is one of the notifiable diseases listed by the World Organization for Animal Health. Occurrence of glanders imposes restriction on equestrian events and restricts equine movement, thus causing economic losses to equine industry. The genetic diversity and global distribution of the causing agent,  Burkholderia mallei, is an obligate pathogen that can be transmitted between animals, but in rare cases also infects human, making glanders a zoonotic disease. However, the main hosts are equids, e.g., horses, donkeys and mules, wherefore glanders has an explicitly strong economic impact in rural areas of regions where B. mallei is endemic, e.g., Brazil, Tukey, Pakistan and India , which can be determined I values –15. ThesB. mallei  deletion; however, this process is still continuing  of various IS families including transposase-coding genes in the genome of . mallei , which antinuing , 6. An iB. mallei, reliable and quick differentiation of this organism from B. pseudomallei is an important step in case of suspected glanders infections. Due to the usually low number of bacteria in infected material , were found to be unreliable due to point mutations in B. pseudomallei sequences which could lead to an incorrect classification of B. pseudomallei as B. mallei in the restriction digestion of the PCR product  recommends the usage of two PCR-assays, a conventional PCR assay by Scholz et al.  and the  element , 22, cau. mallei  21, 22), 22B. ma. mallei , possibln events , 28, vign events , that suommended . For traB. mallei strains has not been studied as extensively as that of other species of the genus Burkholderia , RAPD (random amplified polymorphic DNA), variable number of tandem repeat analysis (MLVA) and multi-locus sequence typing (MLST) were applied so far to B. mallei strains, e.g., out of 120 investigated B. mallei strains, 118 strains were classified as ST40 while merely two strains, NCTC 10247 and NCTC 10260 isolated in Turkey, were identified as ST100  , 5, reveas ST100 . Thus, tose gels , 33, proo groups .B. mallei strains. For example, ribotyping of 25 B. mallei isolates by gDNA digestion using the EcoRI and PstI restriction enzymes followed by labeling with a probed oligonucleotide derived from the ribosomal RNA operon of E. coli revealed 17 distinct ribotypes , that were amplified in a targeted or non-targeted manner, have allowed the identification of distinct ribotypes or profiles within ibotypes , while tpatterns . Again, in India .B. pseudomallei genomes and based on the analysis of PCR amplified tandem repeats of 32 loci (MLVA-32). The initial MLVA typing study conducted on B. mallei included 21 strains from different geographical regions and identified 19 genotypes  corresponding to the variable markers for B. mallei  techniques presented a new opportunity for gaining insight in genomes at an unprecedented resolution. The Illumina sequencing technology can be assumed to be currently the most frequently employed high-throughput genome sequencing method  as it alB. mallei complicate the reconstruction of genomes: short reads cannot span long tandemly arrayed repeats that are longer than the read length, while genomic regions with a high GC content often exhibit low read coverage, primarily caused by PCR bias. These ambiguous genomic regions lead to gaps in the assemblies and thus to fragmented genomes comprising tens or hundreds of contigs .–51.B. maThe lack of contiguity in genome sequences based on Illumina reads can be overcome by sequencing technologies of the so-called third generation, that generate longer reads, like PacBio or nanopore sequencing technologies . Longer B. mallei, WGS not only helped elucidating the genome structure of the pathogen on a fine scale, but was also employed for unambiguously revealing the evolutionary relationship between B. mallei and B. pseudomallei at the genome level. The first B. mallei whole genome sequence was published in 2004 , 99  obvious , althoug obvious . CombiniB. mallei outbreak studies in addition to conventional methods such as MLVA -PCR which detects differences in the melting temperature of PCR amplified products due to single nucleotide polymorphism (SNP) in a target allele has gained attention for molecular epidemiological investigation of bacterial pathogens . It is dB. mallei typing was first introduced by Girault et al. , respectively. Nevertheless, the discriminatory power of both schemes was demonstrated for the global B. mallei diversity as well as for local glanders outbreaks in Bahrain/ Dubai and Pakistan, respectively. By employing cgMLST analysis, geographical clusters of B. mallei could be identified. For example, three different lineages were detected in India, as well as different clusters in Bahrain and Dubai. On the other hand, the B. mallei population in Pakistan proved to be highly homogenic, which reflects the movement of equids between provinces (A different WGS-based typing method that profits from contiguous and correct genome assembly is core genome multi locus sequence typing (cgMLST). A survey investigating the implementation of WGS and thereon-based techniques in European national public health reference laboratories found that cgMLST was the most frequently used typing approach . For thirculosis , 67. Altrculosis , it can rculosis , 69. Regstabilis , 71. Rec. mallei , 59. Botrovinces , 59. Approvinces  additionA drawback of cgMLST analysis is the necessity of assembled genomes as basis for the analysis. Although Appelt et al.  state thB. pseudomallei it was shown that SNP typing provided a better phylogenetic resolution than MLST by revealing that identical MLST types from Cambodia and Australia were caused by homoplasy  that exhibited a certain connection between genotype and geographic distribution: strains from Turkey were found to be highly similar, while strains from India were present in two lineages and altogether three branches. Likewise, two strains from Iran were assigned to different lineages. Due to the lack of available data, only 45 strains were included in this analysis, wherefore it can be supposed that a high proportion of the B. mallei genetic diversity remains to be detected. However, when comparing the clustering patterns generated by PCR-based MLVA and WGS-based SNP analysis, discrepancies become apparent as both lineages that were identified by SNP analysis intermingle in the MLVA results.Few studies employed WGS-based SNP typing to k events , 63, attk events  providedB. mallei strains, which both methods clustered identically  that improve the sequence accuracy of nanopore reads are developed  and alsoShigella sp. from the closely related E. coli (B. mallei, analysis of genome data already has contributed to the improvement of conventional typing methods by revealing new phylogenetically informative SNPs that were incorporated in HRM-PCR analysis (B. mallei, which is genetically homogenous and has high congruence with B. pseudomallei, WGS approaches might offer the possibility to develop assays with high specificity. However, a large set of diverse high-quality assemblies is required for ensuring the reliability of newly identified genomic targets and the targets have to be thoroughly validated in order to prevent false negative or false positive results (In other pathogens, genome data is employed for the development of more specific DNA-based diagnostics, e.g., the development of a robust, highly specific PCR assay for differentiation of  E. coli . Regardianalysis . Especia results .B. mallei is of special public concern as it could potentially be used in terrorist attacks. Thus, the possibility of the occurrence of genetically modified strains has to be considered. WGS would enable the quick detection of genetic manipulation leading to a gain of function, e.g., artificially introduced genes that increase virulence or pathogenicity. Further, the natural gain of resistances by mutations in housekeeping genes and the spread of genes can be detected and monitored by WGS, either based on assemblies or even on sequence reads (B. mallei, yet. There is a lack of studies on the connection between genomic features and phenotypic traits, that has to be addressed in future studies. Apart from genotyping, sequencing may allow insight in gene regulation and the prediction of connections between processes, e.g., by transcriptome sequencing, like quorum sensing in Burkolderia sp. (Being a biothreat agent of the category B  and Tierce reads , 79, 80.eria sp. .B. mallei sequences prevents comprehensive investigation of the global B. mallei genotype distribution as well as tracing of infection sources. This is not caused by a lack of awareness for the disease. In fact, most countries have strict regulations and control measure for preventing glanders outbreaks and the disease is listed in the WOAH Terrestrial Animal Health Code, meaning member states have to report cases to the organization. Nevertheless, isolation of strains is often not done or not successful due to a low bacterial load in clinical specimens (B. mallei strains are not systematically sequenced and if they are, sequence data is not systematically deposited in public databases and thus unavailable for comprehensive analyses. Likewise, incorrect database entries regarding strain identity and metadata have to be corrected. Appelt et al. (Likewise, the lack of publicly available pecimens . B. mallt et al.  identifit et al. . This alB. mallei global genotype distribution.To avoid the problems connected to isolation of bacteria, obtaining sequencing data directly from the sample material by employing a metagenomic approach could offer new possibilities in the future. A higher number and diversity of available sequences will allow a more detailed genotyping, as the choice of reference genome is of utmost importance for detection of informative SNPs. It is advised that instead of using a general reference genome, more detailed analysis can be enabled by utilizing a closely related strain as reference . Thus, iHS conceived the project. HB and ME coordinated the project, wrote the draft version of the manuscript, and prepared the figures. KL and HS also contributed to the text and revised the draft version. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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