Integrated transcriptomic and metabolomic analysis reveals the metabolic programming of GM-CSF- and M-CSF- differentiated mouse macrophages.

Macrophages play a critical role in the inflammatory response and tumor development. Macrophages are primarily divided into pro-inflammatory M1-like and anti-inflammatory M2-like macrophages based on their activation status and functions. In vitro macrophage models could be derived from mouse bone marrow cells stimulated with two types of differentiation factors: GM-CSF (GM-BMDMs) and M-CSF (M-BMDMs), to represent M1- and M2-like macrophages, respectively. Since macrophage differentiation requires coordinated metabolic reprogramming and transcriptional rewiring in order to fulfill their distinct roles, we combined both transcriptome and metabolome analysis, coupled with experimental validation, to gain insight into the metabolic status of GM- and M-BMDMs. The data revealed higher levels of the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and urea and ornithine production from arginine in GM-BMDMs, and a preference for glycolysis, fatty acid storage, bile acid metabolism, and citrulline and nitric oxide (NO) production from arginine in M-BMDMs. Correlation analysis with the proteomic data showed high consistency in the mRNA and protein levels of metabolic genes. Similar results were also obtained when compared to RNA-seq data of human monocyte derived macrophages from the GEO database. Furthermore, canonical macrophage functions such as inflammatory response and phagocytosis were tightly associated with the representative metabolic pathways. In the current study, we identified the core metabolites, metabolic genes, and functional terms of the two distinct mouse macrophage populations. We also distinguished the metabolic influences of the differentiation factors GM-CSF and M-CSF, and wish to provide valuable information for in vitro macrophage studies.

Bioactive compound C498-0670 alleviates LPS-induced sepsis via JAK/STAT and NFκB signaling pathways.

The JAK/STAT and NFκB signaling pathways are two major inflammatory signaling pathways that are usually activated simultaneously in the body's inflammatory response to bacterial or viral infections. Hyperactivation of these two prominent signaling pathways is associated with various immune-related diseases and mortality, pointing to an urgent need for drug development targeting JAK/STAT and/or NFκB signaling. In this study, we screened 18,840 compounds using our well-established dual STAT-NFκB driven luciferase reporter based high-throughput screening system and identified a bioactive compound C498-0670, which inhibits both JAK/STAT and NFκB signaling. C498-0670 inhibits the activation of STATs and p-IKKα/ß in both the immortalized cell lines and primary peritoneal macrophages, while suppressing the expression of LPS-induced inflammatory mediators in vitro. In addition, the overall anti-inflammatory effects of C498-0670 were investigated using transcriptome sequencing and bioinformatics approaches. C498-0670 was predicted to alleviate sepsis/septic shock by disease/function analysis using IPA software, which was further verified in the LPS-induced mouse sepsis model in vivo. C498 reduced LPS-induced liver and kidney damage, myeloid cell infiltration, and pro-inflammatory cytokine and chemokine production in vivo. Furthermore, the SPR-HPLC-MS-based target fishing approach was used to identify the putative drug targets, and the high affinities of JAK2 (JAK/STAT signaling), NFKBIA (NFκB signaling), and IL-1ß, NLRP1b (inflammasome signaling) for C498-0670 were verified by molecular docking approach. These results suggest that C498-0670 can be used as a dual-target inhibitor of JAK/STAT and NFκB signaling pathways for the treatment of various inflammatory diseases, especially septic shock.

STX5 Inhibits Hepatocellular Carcinoma Adhesion and Promotes Metastasis by Regulating the PI3K/mTOR Pathway.

Background and Aims: Syntaxin 5 (STX5) is a member of the syntaxin or target-soluble SNAP receptor (t-SNARE) family and plays a critical role in autophagy. However, its function and molecular mechanism in tumor cell migration are still unknown. The role of STX5 in influencing hepatocellular carcinoma (HCC) is an important topic in our research. Methods: By using quantitative reverse transcription polymerase chain reaction (qPCR), western blotting, and immunohistochemical analysis of RNA and protein in tissues, we comprehensively evaluated data sets from public databases and clinical patient cohorts for STX5. The correlation of STX5 expression with the clinicopathological characteristics of HCC patients were assessed. In addition, we predicted signal pathways from differentially expressed genes (DEGs) and the Cancer Genome Atlas (TCGA) databases, and confirmed the prediction using integrated transcriptome and RNA-seq. We further investigated the underlying mechanisms of STX5 in the migration and adhesion of HCC cells both in vitro and in vivo. Results: In the TCGA dataset and our patient cohort, STX5 levels were significantly higher in HCC tissues than in adjacent normal liver tissues. At the same time, high expression of STX5 predicted worse prognosis in patients with liver cancer. High expression of STX5 indicates the decrease of adhesion and the increase of migration of HCC cells, and the conversion of epithelial-mesenchymal transition (EMT) in vitro via PI3K/mTOR pathway activation. Conversely, when Sirolimus, a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) inhibitor acts on cells simultaneously, STX5 overexpression-mediated enhancement of HCC metastasis is reversed. Double-negative regulation of STX5 and mTOR further enhanced the inhibitory effect of STX5 on HCC metastasis. In vivo, STX5 knockdown inhibited the metastasis of HCC cells. Conclusions: Our study demonstrates a novel research result that STX5 promotes HCC metastasis through PI3K/mTOR pathway. We believe that combined inhibition of STX5 and mTOR is a potential treatment for effectively prolonging patient survival and inhibiting HCC metastasis.

Type I interferon signaling facilitates resolution of acute liver injury by priming macrophage polarization.

Due to their broad functional plasticity, myeloid cells contribute to both liver injury and recovery during acetaminophen overdose-induced acute liver injury (APAP-ALI). A comprehensive understanding of cellular diversity and intercellular crosstalk is essential to elucidate the mechanisms and to develop therapeutic strategies for APAP-ALI treatment. Here, we identified the function of IFN-I in the myeloid compartment during APAP-ALI. Utilizing single-cell RNA sequencing, we characterized the cellular atlas and dynamic progression of liver CD11b+ cells post APAP-ALI in WT and STAT2 T403A mice, which was further validated by immunofluorescence staining, bulk RNA-seq, and functional experiments in vitro and in vivo. We identified IFN-I-dependent transcriptional programs in a three-way communication pathway that involved IFN-I synthesis in intermediate restorative macrophages, leading to CSF-1 production in aging neutrophils that ultimately enabled Trem2+ restorative macrophage maturation, contributing to efficient liver repair. Overall, we uncovered the heterogeneity of hepatic myeloid cells in APAP-ALI at single-cell resolution and the therapeutic potential of IFN-I in the treatment of APAP-ALI.

Effects of Residual Composition and Distribution on the Structural Characteristics of the Protein.

The effect of ratio and consecutive number of hydrophobic residues in the repeating unit of protein chains was investigated by MD simulation. The modified off-lattice HNP model was applied in this study. The protein chains constituted by different HNP ratios or different numbers of consecutively hydrophobic residues with the same chain length were simulated under a broad temperature range. We concluded that the proteins with higher ratio or larger number of sequentially hydrophobic residues present more orientated and compact structure under a certain low temperature. It is attributed to the lower non-bonded potential energy between H-H residual pairs, especially more hydrophobic residues in a procession among the protein chain. Considering the microscopic structure of the protein, more residue contacts are achieved with the proteins with higher ratios and sequential H residues under the low temperature. Meanwhile, with the ratio and consecutive number of H residues increasing, the distribution of stem length showed a transition from exponential decline to unimodal and even multiple peaks, indicating the specific ordered structure formed. These results provide an insight into 3D structural properties of proteins from their residue sequences, which has a primary structure at molecular level and, ultimately, a practical possibility of applying in biotechnological applications.

Targeted therapeutics and novel signaling pathways in non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH).

Non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH) has become the leading cause of liver disease worldwide. NASH, an advanced form of NAFL, can be progressive and more susceptible to developing cirrhosis and hepatocellular carcinoma. Currently, lifestyle interventions are the most essential and effective strategies for preventing and controlling NAFL without the development of fibrosis. While there are still limited appropriate drugs specifically to treat NAFL/NASH, growing progress is being seen in elucidating the pathogenesis and identifying therapeutic targets. In this review, we discussed recent developments in etiology and prospective therapeutic targets, as well as pharmacological candidates in pre/clinical trials and patents, with a focus on diabetes, hepatic lipid metabolism, inflammation, and fibrosis. Importantly, growing evidence elucidates that the disruption of the gut-liver axis and microbe-derived metabolites drive the pathogenesis of NAFL/NASH. Extracellular vesicles (EVs) act as a signaling mediator, resulting in lipid accumulation, macrophage and hepatic stellate cell activation, further promoting inflammation and liver fibrosis progression during the development of NAFL/NASH. Targeting gut microbiota or EVs may serve as new strategies for the treatment of NAFL/NASH. Finally, other mechanisms, such as cell therapy and genetic approaches, also have enormous therapeutic potential. Incorporating drugs with different mechanisms and personalized medicine may improve the efficacy to better benefit patients with NAFL/NASH.

Identifying gene variants underlying the pathogenesis of diabetic retinopathy based on integrated genomic and transcriptomic analysis of clinical extreme phenotypes.

Diabetic retinopathy (DR) is a common complication and the leading cause of blindness in patients with type 2 diabetes. DR has been shown to be closely correlated with blood glucose levels and the duration of diabetes. However, the onset and progression of DR also display clinical heterogeneity. We applied whole-exome sequencing and RNA-seq approaches to study the gene mutation and transcription profiles in three groups of diabetic patients with extreme clinical phenotypes in DR onset, timing, and disease progression, aiming to identify genetic variants that may play roles in the pathogenesis of DR. We identified 23 putatively pathogenic genes, and ingenuity pathway analysis of these mutated genes reveals their functional association with glucose metabolism, diabetic complications, neural system activity, and dysregulated immune responses. In addition, ten potentially protective genes were also proposed. These findings shed light on the mechanisms underlying the pathogenesis of DR and may provide potential targets for developing new strategies to combat DR.

The Culture Dish Surface Influences the Phenotype and Dissociation Strategy in Distinct Mouse Macrophage Populations.

The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity of harvested macrophages. Several previous studies assessed the detachment efficacies of enzymatic and non-enzymatic methods for harvesting the single cell suspensions of macrophages, but a comprehensive study assessing different dissociation methods and culture conditions for detaching functionally different macrophage populations has not yet been reported. In this study, via the well-established GM-CSF and M-CSF differentiated bone marrow derived macrophage models (GM-BMDMs and M-BMDMs), we compared four commonly used enzymatic (trypsin and accutase) and non-enzymatic (PBS and EDTA) dissociation methods along with necessary mechanical detaching steps (scraping and pipetting) to evaluate the viable cell recovery and cell surface marker integrality of GM-BMDMs and M-BMDMs cultured on standard cell culture dish (TC dish), or on culture dish (noTC dish) that was not conditioned to enhance adherence. The data showed that accutase yielded a better recovery of viable cells comparing with PBS and EDTA, especially for tightly adherent GM-BMDMs on TC dishes, with a relatively higher level of detected cell membrane marker F4/80 than trypsin. An additional gradient centrifugation-based dead cell removal approach could increase the proportion of viable cells for TC cultured GM-BMDMs after accutase dissociation. Furthermore, transcriptome analysis was performed to evaluate the putative influence of culture dishes. At steady state, BMDMs cultured on noTC dishes exhibited more proinflammatory gene expression signatures (e.g. IL6, CXCL2 and ILlß) and functions (e.g. TNF and IL17 signaling pathways). Similar inflammatory responses were observed upon LPS challenge regardless of culture conditions and differentiation factors. However, in LPS treated samples, the difference of gene expression patterns, signaling pathways and molecular functions between TC and noTC cultured BMDMs were largely dependent on the types of growth factors (M-CSF and GM-CSF). This observation might provide valuable information for in vitro macrophage studies.

Association Between Metabolic Syndrome and Risk of Renal Cell Cancer: A Meta-Analysis.

Background: Metabolic syndrome (MetS) has been related to increased risks of a variety of cancers. However, the association between MetS and the risk of renal cell cancer (RCC) remains not fully determined. This meta-analysis was conducted to investigate whether MetS is independently associated with the risk of RCC in adults. Methods: Relevant observational studies were obtained by searching PubMed, Embase, Cochrane's Library, and Web of Science databases. Study characteristics and outcome data were extracted independently by two authors. The random-effect model was used for meta-analysis considering the possible influence of between-study heterogeneity. Predefined subgroup analyses were used to evaluate the possible influences of study characteristics on the outcome. Results: Eight studies involving 10,601,006 participants contributed to the meta-analysis. Results showed that MetS was independently associated with a higher risk of RCC in adult population (risk ratio [RR]: 1.62, 95% confidence interval [CI]: 1.41 to 1.87, p<0.001; I2 = 85%). Subgroup analyses showed consistent association in men (RR: 1.52, 95% CI: 1.23 to 1.89, p<0.001) and in women (RR: 1.71, 95% CI: 1.28 to 2.27, p<0.001), in Asians (RR: 1.51, 95% CI: 1.25 to 1.83, p<0.001) and in Caucasians (RR: 1.76, 95% CI: 1.46 to 2.12, p<0.001), and in community derived (RR: 1.56, 95% CI: 1.34 to 1.82, p<0.001) and non-community derived population (RR: 1.87, 95% CI: 1.71 to 2.04, p<0.001). Differences in study design or quality score also did not significantly affect the association (p for subgroup difference both >0.05). Conclusions: MetS may be independently associated with RCC in adult population.

Transmissible ER stress between macrophages and tumor cells configures tumor microenvironment.

Endoplasmic reticulum (ER) stress initiates the unfolded protein response (UPR) and is decisive for tumor cell growth and tumor microenvironment (TME) maintenance. Tumor cells persistently undergo ER stress and could transmit it to the neighboring macrophages and surroundings. Tumor infiltrating macrophages can also adapt to the microenvironment variations to fulfill their highly energy-demanding and biological functions via ER stress. However, whether the different macrophage populations differentially sense ER stress and transmit ER stress to surrounding tumor cells has not yet been elucidated. Here, we aimed to investigate the role of transmissible ER stress, a novel regulator of intercellular communication in the TME. Murine bone marrow-derived macrophage (BMDM) can be polarized toward distinct functional endpoints termed classical (M1) and alternative (M2) activation, and their polarization status has been shown to be tightly correlated with their functional significance. We showed that tumor cells could receive the transmissible ER stress from two differentially polarized macrophage populations with different extent of ER stress activation. The proinflammatory M1-like macrophages respond to ER stress with less extent, however they could transmit more ER stress to tumor cells. Moreover, by analyzing the secreted components of two ER-stressed macrophage populations, we identified certain damage-associated molecular patterns (DAMPs), including S100A8 and S100A9, which are dominantly secreted by M1-like macrophages could lead to significant recipient tumor cells death in synergy with transferred ER stress.

Inhibition of Histone H3 Lysine-27 Demethylase Activity Relieves Rheumatoid Arthritis Symptoms via Repression of IL6 Transcription in Macrophages.

Rheumatoid arthritis (RA) occurs in about 5 per 1,000 people and can lead to severe joint damage and disability. However, the knowledge of pathogenesis and treatment for RA remains limited. Here, we found that histone demethylase inhibitor GSK-J4 relieved collagen induced arthritis (CIA) symptom in experimental mice model, and the underlying mechanism is related to epigenetic transcriptional regulation in macrophages. The role of epigenetic regulation has been introduced in the process of macrophage polarization and the pathogenesis of inflammatory diseases. As a repressive epigenetic marker, tri-methylation of lysine 27 on histone H3 (H3K27me3) was shown to be important for transcriptional gene expression regulation. Here, we comprehensively analyzed H3K27me3 binding promoter and corresponding genes function by RNA sequencing in two differentially polarized macrophage populations. The results revealed that H3K27me3 binds on the promoter regions of multiple critical cytokine genes and suppressed their transcription, such as IL6, specifically in M-CSF derived macrophages but not GM-CSF derived counterparts. Our results may provide a new approach for the treatment of inflammatory and autoimmune disorders.

The phagocytic receptors of ß-glucan.

Phagocytosis is a cellular process maintaining tissue balance and plays an essential role in initiating the innate immune response. The process of phagocytosis was triggered by the binding of pathogen-associated molecular patterns (PAMP) with their cell surface receptors on the phagocytes. These receptors not only perform phagocytic functions, but also bridge the gap between extracellular and intracellular communication, leading to signal transduction and the production of inflammatory mediators, which are crucial for clearing the invading pathogens and maintaining cell homeostasis. For the past few years, the application of ß-glucan comes down to immunoregulation and anti-tumor territory. As a well-known PAMP, ß-glucan is one of the most abundant polysaccharides in nature. By binding to specific receptors on immune cells and activating intracellular signal transduction pathways, it causes phagocytosis and promotes the release of cytokines. Further retrieval and straightening out literature related to ß-glucan phagocytic receptors will help better elucidate their immunomodulatory functions. This review attempts to summarize physicochemical properties and specific processes involved in ß-glucan induced phagocytosis, its phagocytic receptors, and cascade events triggered by ß-glucan at the cellular and molecular levels.

C644-0303, a small-molecule inhibitor of the Wnt/ß-catenin pathway, suppresses colorectal cancer growth.

The Wnt/ß-catenin signaling pathway plays an important role in tissue homeostasis, and its malignant activation is closely related to the occurrence and development of many cancers, especially colorectal cancer with adenomatous polyposis coli (APC) and CTNNB1 mutations. By applying a TCF/lymphoid-enhancing factor (LEF) luciferase reporter system, the high-throughput screening of 18 840 small-molecule compounds was performed. A novel scaffold compound, C644-0303, was identified as a Wnt/ß-catenin signaling inhibitor and exhibited antitumor efficacy. It inhibited both constitutive and ligand activated Wnt signals and its downstream gene expression. Functional studies showed that C644-0303 causes cell cycle arrest, induces apoptosis, and inhibits cancer cell migration. Moreover, transcription factor array indicated that C644-0303 could suppress various tumor-promoting transcription factor activities in addition to Wnt/ß-catenin. Finally, C644-0303 suppressed tumor spheroidization in a 3-dimensional cell culture model and inhibited xenograft tumor growth in mice. In conclusion, we report a novel structural small molecular inhibitor targeting the Wnt/ß-catenin signaling pathway that has therapeutic potential for colorectal cancer treatment.

Scaffold compound L971 exhibits anti-inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways.

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune-related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high-throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli-dependent activation of STAT1, STAT3 and IκBα and could significantly down-regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high-throughput RNA sequencing, and significant differentially up-regulated and down-regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti-inflammatory effects of L971. Finally, L971 anti-inflammatory character was further verified in LPS-induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down-regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.

Small-Molecule Inhibitors Targeting the Canonical WNT Signaling Pathway for the Treatment of Cancer.

Canonical WNT signaling is an important developmental pathway that has attracted increased attention for anticancer drug discovery. From the production and secretion of WNT ligands, their binding to membrane receptors, and the ß-catenin destruction complex to the expansive ß-catenin transcriptional complex, multiple components have been investigated as drug targets to modulate WNT signaling. Significant progress in developing WNT inhibitors such as porcupine inhibitors, tankyrase inhibitors, ß-catenin/coactivators, protein-protein interaction inhibitors, casein kinase modulators, DVL inhibitors, and dCTPP1 inhibitors has been made, with several candidates (e.g., LGK-974, PRI-724, and ETC-159) in human clinical trials. Herein we summarize recent progress in the drug discovery and development of small-molecule inhibitors targeting the canonical WNT pathway, focusing on their specific target proteins, in vitro and in vivo activities, physicochemical properties, and therapeutic potential. The relevant opportunities and challenges toward maintaining the balance between efficacy and toxicity in effectively targeting this pathway are also highlighted.

Short-term exposure to air pollution and occurrence of emergency stroke in Chongqing, China.

OBJECTIVE: This study aimed to study the relationship between air pollution and stroke (especially emergency stroke) in different regions and determine which air pollutant is the most significantly associated with stroke. METHODS: The number of patients with emergency stroke, air pollutant data and related meteorological indicators were collected from December 2013 to May 2018 for large comprehensive hospitals in Chongqing. The generalized additive model was used to analyse the relationship between air pollution and emergency stroke. RESULTS: After analysis and adjusting for meteorological indicators and day-of-the-week effects, in the one-pollutant model, every 10 µg/m3 increase in ozone(O3) was associated with a 2.482% (95% CI 1.044%, 3.919%) change in emergency strokes within lag0. For males, every 10 µg/m3 increase of O3 contributed to a 0.77% percent greater change compared with females. For the group younger than 60 years, we observed a 1.14% increase in risk with every 10 µg/m3 increase in O3. The group with pre-existing hypertension had a 0.26% higher risk than the group with no pre-existing hypertension with every 10 µg/m3 increase in O3. In two-pollutant model, when O3 was combined with a 10 µg/m3 increase of NO2, it increased the most significant risk of emergency stroke by 0.22%. CONCLUSION: These findings suggest that short-term exposure to O3 within 0 days is associated with emergency outpatient strokes, and younger people (age < 60 years) males and people with hypertension are more sensitive than older people, females and people without pre-existing hypertension.

A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses.

Type I interferons (IFN-I) protect us from viral infections. Signal transducer and activator of transcription 2 (STAT2) is a key component of interferon-stimulated gene factor 3 (ISGF3), which drives gene expression in response to IFN-I. Using electron microscopy, we found that, in naive cells, U-STAT2, lacking the activating tyrosine phosphorylation, forms a heterodimer with U-STAT1 in an inactive, anti-parallel conformation. A novel phosphorylation of STAT2 on T404 promotes IFN-I signaling by disrupting the U-STAT1-U-STAT2 dimer, facilitating the tyrosine phosphorylation of STATs 1 and 2 and enhancing the DNA-binding ability of ISGF3. IKK-ε, activated by virus infection, phosphorylates T404 directly. Mice with a T-A mutation at the corresponding residue (T403) are highly susceptible to virus infections. We conclude that T404 phosphorylation drives a critical conformational switch that, by boosting the response to IFN-I in infected cells, enables a swift and efficient antiviral defense.

Zelnorm, an agonist of 5-Hydroxytryptamine 4-receptor, acts as a potential antitumor drug by targeting JAK/STAT3 signaling.

The Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays central roles in cancer cell growth and survival. Drug repurposing strategies have provided a valuable approach for developing antitumor drugs. Zelnorm (tegaserod maleate) was originally designed as an agonist of 5-hydroxytryptamine 4 receptor (5-HT4R) and approved by the FDA for treating irritable bowel syndrome with constipation (IBS-C). Through the use of a high-throughput drug screening system, Zelnorm was identified as a JAK/STAT3 signaling inhibitor. Moreover, the inhibition of STAT3 phosphorylation by Zelnorm was independent of its original target 5-HT4R. Zelnorm could cause G1 cell cycle arrest, induce cell apoptosis and inhibit the growth of a variety of cancer cells. The present study identifies Zelnorm as a novel JAK/STAT3 signaling inhibitor and reveals a new clinical application of Zelnorm upon market reintroduction.

Discovery and evaluation of Atopaxar hydrobromide, a novel JAK1 and JAK2 inhibitor, selectively induces apoptosis of cancer cells with constitutively activated STAT3.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway plays a vital role in immunity, cell division, cell death and tumor formation. Disrupted JAK-STAT signaling may lead to various diseases, especially cancer and immune disorders. Because of its importance, this signaling pathway has received significant attention from the pharmaceutical and biotechnology industries as a therapeutic target for drug design. However, few JAK or STATs inhibitors have been developed for cancer treatment. We used an in vitro STAT3 luciferase reporter assay to find novel inhibitors that could effectively block the JAK-STAT pathway. In our study, we screened 16,081 drug-like chemicals and found that atopaxar hydrobromide (AHB) is a specific inhibitor of JAK-STAT3 signaling. Our results suggest that AHB not only blocks constitutively activated and cytokine-induced STAT3 phosphorylation but also inhibits JAK1 and JAK2 phosphorylation. Moreover, AHB induces G1 phase cell cycle arrest, which stops cancer cell growth and induces apoptosis. AHB also inhibited tumor cell growth in vivo. In conclusion, AHB is a potential inhibitor that could be developed as a JAK-STAT pathway drug.