STAT Proteins Research Articles

Evaluation of Genetic or Cellular Impairments in Type I IFN Immunity in a Cohort of Young Adults with Critical COVID-19

Several genetic and immunological risk factors for severe COVID-19 have been identified, with monogenic conditions relating to 13 genes of type I interferon (IFN) immunity proposed to explain 4.8% of critical cases. However, previous cohorts have been clinically heterogeneous and were not subjected to thorough genetic and immunological analyses. We therefore aimed to systematically investigate the prevalence of rare genetic variants causing inborn errors of immunity (IEI) and functionally interrogate the type I IFN pathway in young adults that suffered from critical COVID-19 yet lacked comorbidities. We selected and clinically characterized a cohort of 38 previously healthy individuals under 50 years of age who were treated in intensive care units due to critical COVID-19. Blood samples were collected after convalescence. Two patients had IFN-α autoantibodies. Genome sequencing revealed very rare variants in the type I IFN pathway in 31.6% of the patients, which was similar to controls. Analyses of cryopreserved leukocytes did not indicate any defect in plasmacytoid dendritic cell sensing of TLR7 and TLR9 agonists in patients carrying variants in these pathways. However, lymphocyte STAT phosphorylation and protein upregulation upon IFN-α stimulation revealed three possible cases of impaired type I IFN signaling in carriers of rare variants. Together, our results suggest a strategy of functional screening followed by genome analyses and biochemical validation to uncover undiagnosed causes of critical COVID-19.

Baricitinib relieves DSS-induced ulcerative colitis in mice by suppressing the NF-κB and JAK2/STAT3 signalling pathways.

Ulcerative colitis (UC) is a relapsing inflammatory disease with a unique aetiology. The treatment of UC is challenging, and the current clinical therapeutics for colitis have limited efficacy. Thus, finding new and effective treatment options remains urgent. Baricitinib, an inhibitor of Janus kinase (JAK), has been clinically used to treat rheumatoid arthritis (RA). However, its potential effects on UC have not been fully elucidated. In this study, we aimed to explore the effects of baricitinib on UC and its underlying mechanism. Dextran sulphate sodium (DSS)-induced murine model of chronic colitis was used to investigate the intervention efficacy following oral administration of baricitinib. The levels of key cytokines, such as IL-6, IFN-γ and IL-17A, were determined. Moreover, western blotting for IκBα, p-IκBα, JAK2, p-JAK2, STAT3 and p-STAT3 protein expression was performed to investigate the associated signalling pathways. Our findings demonstrated that baricitinib can significantly relieve DSS-induced UC in mice. After baricitinib intervention, IL-6, IFN-γ and IL-17A levels were decreased both in vitro and in vivo. Moreover, the elevated expression levels of p-IκBα, p-JAK2, and p-STAT3 were significantly reduced after treatment. Collectively, these results suggest that baricitinib is a potential therapeutic agent for alleviation of DSS-induced colitis. This study provides a method for subsequent investigations on potential curative drugs development of the for colitis.

TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.

Trophoblast-derived miR-410-5p induces M2 macrophage polarization and mediates immunotolerance at the fetal-maternal interface by targeting the STAT1 signaling pathway

BackgroundMacrophages phenotypic deviation and immune imbalance play vital roles in pregnancy-associated diseases such as spontaneous miscarriage. Trophoblasts regulate phenotypic changes in macrophages, however, their underlying mechanism during pregnancy remains unclear. Therefore, this study aimed to elucidate the potential function of trophoblast-derived miRNAs (miR-410-5p) in macrophage polarization during pregnancy.MethodsPatient decidual macrophage tissue samples in spontaneous abortion group and normal pregnancy group (those who had induced abortion for non-medical reasons) were collected at the Reproductive Medicine Center of Renmin Hospital of Wuhan University from April to December 2021. Furthermore, placental villi and decidua tissue samples were collected from patients who had experienced a spontaneous miscarriage and normal pregnant women for validation and subsequent experiments at the Shenzhen Zhongshan Obstetrics & Gynecology Hospital (formerly Shenzhen Zhongshan Urology Hospital), from March 2021 to September 2022. As an animal model, 36 female mice were randomly divided into six groups as follows: naive-control, lipopolysaccharide-model, agomir-negative control prevention, agomir-410-5p prevention, agomir-negative control treatment, and agomir-410-5p treatment groups. We analyzed the miR-410-5p expression in abortion tissue and plasma samples; and supplemented miR-410-5p to evaluate embryonic absorption in vivo. The main source of miR-410-5p at the maternal–fetal interface was analyzed, and the possible target gene, signal transducer and activator of transcription (STAT) 1, of miR-410-5p was predicted. The effect of miR-410-5p and STAT1 regulation on macrophage phenotype, oxidative metabolism, and mitochondrial membrane potential was analyzed in vitro.ResultsMiR-410-5p levels were lower in the spontaneous abortion group compared with the normal pregnancy group, and plasma miR-410-5p levels could predict pregnancy and spontaneous abortion. Prophylactic supplementation of miR-410-5p in pregnant mice reduced lipopolysaccharide-mediated embryonic absorption and downregulated the decidual macrophage pro-inflammatory phenotype. MiR-410-5p were mainly distributed in villi, and trophoblasts secreted exosomes-miR-410-5p at the maternal–fetal interface. After macrophages captured exosomes, the cells shifted to the tolerance phenotype. STAT1 was a potential target gene of miR-410-5p. MiR-410-5p bound to STAT1 mRNA, and inhibited the expression of STAT1 protein. STAT1 can drive macrophages to mature to a pro-inflammatory phenotype. MiR-410-5p competitive silencing of STAT1 can avoid macrophage immune disorders.ConclusionMiR-410-5p promotes M2 macrophage polarization by inhibiting STAT1, thus ensuring a healthy pregnancy. These findings are of great significance for diagnosing and preventing spontaneous miscarriage, providing a new perspective for further research in this field.

Molecular insights into STAT1a protein in rohu (Labeo rohita): unveiling expression profiles, SRC homology domain recognition, and protein-protein interactions triggered by poly I: C.

STAT1a is an essential signal transduction protein involved in the interferon pathway, playing a vital role in IFN-alpha/beta and gamma signaling. Limited information is available about the STAT protein in fish, particularly in Indian major carps (IMC). This study aimed to identify and characterize the STAT1a protein in Labeo rohita (LrSTAT1a). The full-length CDS of LrSTAT1a transcript was identified and sequenced. Phylogenetic analyses were performed based on the nucleotide sequences. The in-vivo immune stimulant poly I: C was used to treat various tissues, and the expression of LrSTAT1a was determined using quantitative real-time polymerase chain reaction (qRT-PCR). A 3D model of the STAT1a protein was generated using close structure homologs available in the database and checked using molecular dynamics (MD) simulations. The full-length CDS of Labeo rohita STAT1a (LrSTAT1a) transcript consisted of 3238 bp that encoded a polypeptide of 721 amino acids sequence was identified. Phylogenetic analyses were performed based on the nucleotide sequences. Based on our findings, other vertebrates share a high degree of conservation with STAT1a. Additionally, we report that the in vivo immune stimulant poly I: C treatment of various tissues resulted in the expression of LrSTAT1a as determined by quantitative real-time polymerase chain reaction (qRT-PCR). In the current investigation, treatment with poly I: C dramatically increased the expression of LrSTAT1a in nearly every organ and tissue, with the brain, muscle, kidney, and intestine showing the highest levels of expression compared to the control. We made a 3D model of the STAT1a protein by using close structure homologs that were already available in the database. The model was then checked using molecular dynamics (MD) simulations. Consistent with previous research, the MD study highlighted the significance of the STAT1a protein, which is responsible for Src homology 2 (SH2) recognition. An important H-bonding that successfully retains SH2 inside the STAT1a binding cavity was determined to be formed by the conserved residues SER107, GLN530, SER583, LYS584, MET103, and ALA106. This study provides molecular insights into the STAT1a protein in Rohu (Labeo rohita) and highlights the potential role of STAT1a in the innate immune response in fish. The high degree of conservation of STAT1a among other vertebrates suggests its crucial role in the immune response. The in-vivo immune stimulation results indicate that STAT1a is involved in the immune response in various tissues, with the brain, muscle, kidney, and intestine being the most responsive. The 3D model and MD study provide further evidence of the significance of STAT1a in the immune response, specifically in SH2 recognition. Further research is necessary to understand the specific mechanisms involved in the IFN pathway and the role of STAT1a in the immune response of IMC.

Yishen Tongbi decoction attenuates inflammation and bone destruction in rheumatoid arthritis by regulating JAK/STAT3/SOCS3 pathway.

Yishen-Tongbi Decoction (YSTB), a traditional Chinese prescription, has been used to improve syndromes of rheumatoid arthritis (RA) for many years. Previous research has shown that YSTB has anti-inflammatory and analgesic properties. However, the underlying molecular mechanism of the anti-RA effects of YSTB remains unclear. The purpose of this research was to investigate how YSTB affected mice with collagen-induced arthritis (CIA) and RAW264.7 cells induced with lipopolysaccharide (LPS). The findings show that YSTB could significantly improve the clinical arthritic symptoms of CIA mice (mitigate paw swelling, arthritis score, thymus and spleen indices, augment body weight), downregulated expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-17, while upregulated the level of anti-inflammatory like IL-10 and transforming growth factor-β (TGF-β). Meanwhile, YSTB inhibits bone erosion and reduces inflammatory cell infiltration, synovial proliferation, and joint destruction in CIA mice. In addition, we found that YSTB was able to suppress the LPS-induced inflammation of RAW264.7 cells, which was ascribed to the suppression of nitric oxide (NO) production and reactive oxygen species formation (ROS). YSTB also inhibited the production of inducible nitric oxide synthase and reduced the releases of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells. Furthermore, the phosphorylation expression of JAK2, JAK3, STAT3, p38, ERK and p65 protein could be suppressed by YSTB, while the expression of SOCS3 could be activated. Taken together, YSTB possesses anti-inflammatory and prevention bone destruction effects in RA disease by regulating the JAK/STAT3/SOCS3 signaling pathway.

Babao dan inhibits gastric cancer progression in vivo Through multiple signaling pathways

Objective: The aim of this study was to explore the effects of Babao dan (BBD), a traditional Chinese medicine, on gastric cancer (GC) progression in vivo. Materials and Methods: A subcutaneous xenograft mouse model of GC was established using MGC80-3 cells. The terminal deoxynucleotidyl transferase-mediated dUTP: 2'-deoxyuridine 5'-triphosphate -biotin nick-end labeling method was adopted to detect cell apoptosis in vivo. The expression levels of proteins associated with proliferation, apoptosis, and angiogenesis were measured by immunohistochemical staining or western blotting (WB). The activation and protein levels of p-c-Jun N-terminal kinase (JNK), p-p38, p-extracellular-regulated kinase 1/2, p-nuclear factor-κB (NF-κB), and p-STAT3 were examined by Bio-plex and WB. Results: BBD significantly inhibited tumor growth in GC mouse models with no adverse effect on body weight or organ function. It was also found that BBD significantly suppressed the proliferation of GC tumor cells, induced the apoptosis of tumor cells, and inhibited angiogenesis through inactivating with mitogen-activated protein kinase, NF-κB, and STAT3 pathways. Conclusions: BBD exerts suppressive effects on GC tumor growth by regulating multiple pathways in vivo, which may provide a novel treatment option for GC therapy.

Bioinformatics and Molecular Docking Study of Amentoflavone and 3,8-Biapigenin as Inhibitors on Cervical Cancer Proteins

Cervical cancer maintains its second-place ranking for Indonesia's highest number of cancer cases. In 2021, there were 36,633 cases of cervical cancer in Indonesia, with a rising death rate. Commonly, chemotherapy is used to treat cervical cancer and can improve the survival chances of patients, but these therapies imply increased toxicity. Biflavonoid group compounds like amentoflavone and 3,8-Biapigenin have the potential to act as anticancer agents by modulating multiple signaling pathways. This study aims to determine the cervical anticancer potential of amentoflavone and 3,8-Biapigenin based on in silico study. Prediction of anticancer activity in silico using Prediction of Activity Spectra for Active Substances (PASS) online, followed by target protein tracing using STITCH-STRING, then receptor analysis test using Ramachandran plot. A molecular docking test was conducted to determine the binding affinity of the compound with the receptor. Based on the online PASS, the compounds as thought to have low cervical anticancer potential if tested on a laboratory scale. STAT3, EP300, CYP1A1, and AKR1C1 proteins used in this study have met the requirements of a suitable receptor for molecular docking test. The best binding affinity was obtained at the interaction of amentoflavone and STAT3 with a better docking score (-9.3 kcal/mol) than doxorubicin (-7.1 kcal/mol). Overall, the results suggest biflavonoid compounds have the potential to be developed as a chemopreventive agent for cervical cancer.Keywords: bioinformatics, molecular docking, amentoflavone, 3,8-Biapigenin, cervical cancer protein.

Anti-Inflammatory Effects of the LK5 Herbal Complex on LPS- and IL-4/IL-13-Stimulated HaCaT Cells and a DNCB-Induced Animal Model of Atopic Dermatitis in BALB/c Mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease influenced by a complex interplay of genetic and environmental factors. The activation of the JAK-STAT pathway increases the expression of inflammatory cytokines such as IL-4 and IL-13, further deteriorating AD. Therefore, for the treatment of AD, the JAK-STAT pathway is emerging as a significant target, alongside inflammatory cytokines. This study investigates the potential therapeutic effects of a novel herbal complex, LK5, composed of Scutellaria baicalensis, Liriope platyphylla, Sophora flavescens, Dictammus dasycarpus, and Phellodendron schneider, known for their anti-inflammatory and immune-modulating properties. We examined the anti-inflammatory and anti-AD effects of the LK5 herbal complex in HaCaT cells stimulated by LPS and IL-4/IL-13, as well as in a mouse model of AD induced by DNCB. In HaCaT cells stimulated with LPS or IL-4/IL-13, the LK5 herbal complex demonstrated anti-inflammatory effects by inhibiting the expression of inflammatory cytokines including TNF-α, IL-6, and IL-1β, and downregulating the phosphorylation of STAT proteins. In a murine AD-like model induced by DNCB, administration of the LK5 herbal complex significantly ameliorated clinical symptoms, including dermatitis, ear thickness, and TEWL. Histological analysis revealed a reduction in epidermal thickness and mast cell infiltration. The LK5 herbal complex also inhibited pruritus induced by compound 48/80. Furthermore, the LK5 herbal complex treatment significantly decreased the levels of inflammatory cytokines such as TSLP, IL-6, and IgE in plasma and ear tissue of AD-induced mice. These findings suggest that the LK5 herbal complex may modulate the immune response and alleviate AD symptoms by inhibiting STAT pathways.

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Association of STAT4 Gene Polymorphisms (rs10181656, rs7574865, rs7601754, rs10168266) and Serum STAT4 Levels in Age-Related Macular Degeneration.

To investigate the association between STAT4 (rs10181656, rs7574865, rs7601754, and rs10168266) gene polymorphisms and STAT4 serum levels in patients with age-related macular degeneration. The study included 150 individuals with early AMD, 150 individuals with exudative AMD, and 200 healthy subjects. DNA was extracted from peripheral blood leukocytes using the DNA salting-out method, and the genotyping was performed using a real-time polymerase chain reaction (RT-PCR) method. STAT4 serum levels were evaluated using the ELISA method. Statistical analysis was performed using "IBM SPSS "Statistics 29.0" software". The study revealed no statistically significant differences in the distribution of genotypes and alleles for the STAT4 polymorphisms (rs10181656, rs7574865, rs7601754, and rs10168266) between patients with AMD and the control group. Similarly, a gender-based analysis did not yield any significant differences in the genotype or allele frequencies. Age group comparisons also showed no statistically significant variations in the presence of these STAT4 polymorphisms between AMD patients and the control group. However, notably, individuals with exudative AMD displayed lower levels of serum STAT4 in comparison to the control group (median (IQR): 0.118 (0.042) vs. 0.262 (0.385), p = 0.005). Investigating STAT4 gene polymorphisms (rs10181656, rs7574865, rs7601754, and rs10168266) did not reveal a significant association with AMD. However, further analysis demonstrated intriguing findings regarding serum STAT4 levels. Exudative AMD patients with at least one G allele of the STAT4 rs10181656 exhibited significantly lower serum STAT4 levels than the control group subjects (p = 0.011). Similarly, those with at least one T allele of STAT4 rs10168266 had lower serum STAT4 levels compared to the control group subjects (p = 0.039). These results suggest a potential link between specific STAT4 genotypes and serum STAT4 levels in exudative AMD patients, shedding light on a novel aspect of the disease.

JAK1 Is a Novel Target of Tumor- and Invasion-Suppressive microRNA 494-5p in Colorectal Cancer.

MiR-494-5p expression has been suggested to be associated with colorectal cancer (CRC) and its metastases in our previous studies. However, functional investigations on the molecule-mediating actions of this miR in CRC are lacking. In silico analysis in the present study revealed a putative binding sequence within the 3'UTR of JAK1. Overexpression of miR-494-5p in cultured CRC significantly reduced the luciferase activity of a reporter plasmid containing the wild-type JAK1-3'UTR, which was abolished by seed sequence mutation. Furthermore, the overexpression of miR-494-5p in CRC cell lines led to a significant reduction in JAK1 expression, proliferation, in vitro migration, and invasion. These effects were abolished by co-transfection with a specific double-stranded RNA that inhibits endogenous miR-494-5p. Moreover, IL-4-induced migration, invasion, and phosphorylation of JAK1, STAT6, and AKT proteins were reduced after an overexpression of this miR, suggesting that this miR affects one of the most essential pathways in CRC. A Kaplan-Meier plotter analysis revealed that patients with high JAK1 expression show reduced survival. Together, these data suggest that miR-494-5p physically inhibits the expression of JAK1 at the translational level as well as in migration and invasion, supporting the hypothesis of miR-494-5p as an early tumor suppressor and inhibitor of early steps of metastasis in CRC.

Uncovering the pharmacological mechanism of Shou Tai Wan on recurrent spontaneous abortion: A integrated pharmacology strategy-based research

Ethnopharmacological relevanceShou Tai Wan (STW), a traditional Chinese medicine formula, has been historically used for the treatment of recurrent spontaneous abortion (RSA). Despite its long-standing usage, the exact mechanism underlying the therapeutic effects of STW remains unclear in the existing literature. Aims of this studyTo explore the Pharmacological Mechanism of STW on RSA. MethodsA network pharmacological methodology was utilized to predict the active compounds and potential targets of STW, collect the RSA targets and other human proteins of STW, and analyze the STW related networks. The animal experiments were also performed to validate the effect of STW on RSA. ResultsThe results of network analysis showed that STW may regulate PI3K/AKT, MAPK, FoxO signaling pathways and so on. Animal experiment established the RSA model with CBA/J × DBA/2 mice. It was found that STW can reduce the embryo absorption rate of RSA group (p < 0.05) and balance the expression of Th 1/Th2 type cytokines compared with the model group. After 14 days of administration, the decidual and placental tissues were taken and the CD4+ T cells were isolated, and the phosphorylation level of signaling pathway was detected by Springbio720 antibody microarray. This experiment found that STW can significantly up-regulate the phosphorylation levels of STAT3 and STAT6 proteins in the STAT signaling pathway, and down-regulating the phosphorylation level of STAT1 protein. STW also significantly up-regulated the phosphorylation levels of Raf1, A-Raf, Ask1, Mek1, Mek2, JKK1, ERK1, ERK2, c-fos, c-Jun and CREB proteins in the MAPK signaling pathway, and down-regulate the phosphorylation levels of MEK6 and IKKb proteins. Compared with the RSA group, the STW group increased the expression levels of ERK1/2 mRNA and proteins and p-ERK1/2 proteins, and there was a statistical difference (p < 0.05). This is consistent with the chip results. ConclusionSTW may achieve therapeutic effects by interfering with the signaling pathways, biological processes and targets discovered in this study. It provides a new perspective for revealing the immunological mechanism of STW in the treatment of RSA, and also provides a theoretical basis for the clinical use of STW in the treatment of RSA.

The "StemDif Sensor Test": A Straightforward, Non-Invasive Assay to Characterize the Secreted Stemness and/or Differentiation Activities of Tumor-Derived Cancer Cell Lines.

Cancer stem cells are a subpopulation of tumor cells characterized by their ability to self-renew, induce tumors upon engraftment in animals and exhibit strong resistance to chemotherapy and radiotherapy. These cells exhibit numerous characteristics in common with embryonic stem cells, expressing some of their markers, typically absent in non-pathological adult differentiated cells. The aim of this study was to investigate the potential of conditioned media from cancer stem cells to modulate the fate of Leukemia Inhibitory Factor (LIF)-dependent murine embryonic stem cells (mESCs) as a way to obtain a direct readout of the secretome of cancer cells. A functional assay, "the StemDif sensor test", was developed with two types of cancer stem cells derived from grade IV glioblastoma (adult and pediatric) or from gastric adenocarcinoma. We show that conditioned media from the selection of adult but not pediatric Glioma-Inducing Cells (GICs) maintain mESCs' pluripotency in correlation with LIF secretion and activation of STAT3 protein. In contrast, conditioned media from gastric adenocarcinoma cells display LIF-independent stemness and differentiation activities on mESC. Our test stands out for its user-friendly procedures, affordability and straightforward output, positioning it as a pioneering tool for in-depth exploration of cancer stem cell secretome characteristics.

Human STAT1 gain of function with chronic mucocutaneous candidiasis: A comprehensive review for strengthening the connection between bedside observations and laboratory research.

Germline human heterozygous STAT1 gain-of-function (GOF) variants were first discovered a common cause of chronic mucocutaneous candidiasis (CMC) in 2011. Since then, numerous STAT1 GOF variants have been identified. A variety of clinical phenotypes, including fungal, viral, and bacterial infections, endocrine disorders, autoimmunity, malignancy, and aneurysms, have recently been revealed for STAT1 GOF variants, which has led to the expansion of the clinical spectrum associated with STAT1 GOF. Among this broad range of complications, it has been determined that invasive infections, aneurysms, and malignancies are poor prognostic factors for STAT1 GOF. The effectiveness of JAK inhibitors as a therapeutic option has been established, although further investigation of their long-term utility and side effects is needed. In contrast to the advancements in treatment options, the precise molecular mechanism underlying STAT1 GOF remains undetermined. Two primary hypotheses for this mechanism involve impaired STAT1 dephosphorylation and increased STAT1 protein levels, both of which are still controversial. A precise understanding of the molecular mechanism is essential for not only advancing diagnostics but also developing therapeutic interventions. Here, we provide a comprehensive review of STAT1 GOF with the aim of establishing a stronger connection between bedside observations and laboratory research.

Exploring the Mechanism of Chang-An-Kang on Active Ulcerative Colitis Rats Based on NF-κB/STAT3 Signaling Pathway

Objectives The objective of this study was to investigate the therapeutic effect and mechanism of Chang-An-Kang on ulcerative colitis rats. Materials and Methods The rat model of ulcerative colitis induced by 6% glacial acetic acid was established. The corresponding therapeutic drugs were given 24 h after the model was established. The pathological morphology of colon tissue was observed after 21 days of continuous administration. The levels of interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) were measured by ELISA kit. The expression levels of NF-κB and STAT3 proteins in colon tissue were detected by IHC. Results The results showed that the changes in the pathological and biochemical indexes of the rat colon tissue indicate that the model was successfully established. Chang-An-Kang could significantly reduce rat colon tissue damage, decrease the content of IL-6, IL-7, and TNF-α, and increase the content of IL-10. The NF-κB and STAT3 protein expression levels were decreased. Conclusion By increasing the content of anti-inflammatory factor IL-10, decreasing the content of pro-inflammatory factors IL-6 and TNF-α, and inhibiting the expression of NF-κB and STAT3 proteins, Chang-An-Kang can reduce inflammatory infiltration. It has an obvious therapeutic effect on acetic acid ulcerative colitis in rats.

Abstract C052: Effect of ginger extracts on hepatocellular carcinoma cell lines-derived from Caucasian, Asian, and African American patients

Abstract Hepatocellular Carcinoma (HCC) is a highly fatal disease with mortality running parallel to its incidence. For HCC, race/ethnicity plays a vital role in determining incidence, mortality, and survival rates. The incidence of HCC is highest in Asia and Africa. Furthermore, there is a statistically significant increase in incidence and mortality and a decrease in 5-year survival rates in African American (AA)/Black patients compared to non-Hispanic white patients. There is a knowledge gap in our understanding of the molecular mechanism underlying the HCC racial disparity between AA/Black, White, and Asian patients. To understand the underlying cause, we performed bioinformatics on existing gene expression data. We found that type I Interferon (IFN-I) signaling pathway showed statistically significant activation in AA/Black patients compared to white patients. Over 60% of Gen Xers/Millennials are taking a more holistic approach to their diet to prevent chronic diseases, including cancer. We hypothesized that dietary compounds exert anticancer effects on HCC, and because of their anti-inflammatory property, they might modulate the IFN-I signaling pathway. We tested ginger extracts on HCC derived from white (HepG2), Black (Hep3B and O/20), and Asian (Hu7) cancer patients. A dose-response of these extracts was used to determine IC50s on these cell lines using an MTT cell proliferation assay. Activation of INF-1-mediated downstream signaling proteins, including JAK1, TYK2, STAT1, and STAT2 phosphorylation status, was determined on a Western blot analysis using phospho-specific primary antibodies. The expression of Interferon Signaling Genes (ISGs), including Myxovirus resistance gene 1 (MX1), 2′,5′-oligoadenylate synthetase (OAS1), Interferon-alpha inducible protein 6 (IFI6), and Interferon stimulated gene 15 (ISG15) was assayed using a quantitative Real-Time Polymerase Chain Reaction (RT-PCR). Ginger has a significantly (P&amp;lt;0.05) lower IC50s (mg/ml) on cell lines from Black patients (Hep3B=156 ± 3, and O/20=162 ± 3) than cell lines from white (HepG2=176±5) or Asian (Hu7=174 ± 5) patients. Our data show that untreated cells exhibited phosphorylation/activation of IFN-1 downstream mediators, including JAK1, TYK2, STAT1, and STAT2 proteins. Ginger treatment reduced the phosphorylation of these IFN downstream mediators in all HCC cell lines. Furthermore, expression of MX1, ISG15, IF16, and OAS1 was also reduced in all HCC cells lines in a dose-dependent manner; however, the effects on gene expression were more sensitive to a lower concentration of ginger extract in Black patients derived HCC cell lines than other HCC cell lines. Currently, research is underway to identify the active chemical agent in ginger and test it on other patient-derived HCC lines and in an in vivo model. In conclusion, our data suggest that ginger can potentially attenuate IFN-mediated signaling pathways in HCC, and cells from Black HCC patients may be more sensitive to ginger (Funded: P20 CA264068-01). Citation Format: Sadia Kanwal, Eva Davis, Seung Lee, Milton Omar Faison, Devanand Sarkar, Rafat Ali Siddiqui. Effect of ginger extracts on hepatocellular carcinoma cell lines-derived from Caucasian, Asian, and African American patients [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C052.

Design, synthesis and anti-rheumatoid arthritis activity of target TLR4 inhibitors

A series of 1-(2-oxocyclohexyl)butane-1, 3-dione derivatives were designed and synthesized as TLR4 inhibitors by modifying the core structure of the lead compound ((6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) carbamate)). In vitro, compound 3p significantly inhibited the proliferation of rat synovial cells, inhibited the proliferation of LPS-induced RAW264.7 cells, and inhibited TLR4 activity, with IC50 values of 1.21 ± 0.09 μM, 0.73 ± 0.05 μM and 0.43 ± 0.03 μM, respectively, which was superior to the positive control methotrexate. In vivo anti-rheumatoid arthritis evaluation, compound 3p can significantly inhibit the inflammatory factors TNF-α, IL-1β and IL-6, so as to achieve the therapeutic purpose. In the preliminary mechanism study, compound 3p has obvious regulatory effects on the abnormal increase of TLR4, JAK2 and STAT3 protein and gene expression related to inflammatory signaling pathway in RAW264.7 cells. In summary, this study aims to develop more effective candidates for rheumatoid arthritis.

Targeting CDK8/CDK19 to Disrupt Leukemic Stem Cell-like Population in Acute Myeloid Leukemia: Exploring RVU120 As a Promising Frontline Therapy

Leukemic stem cells (LSCs) are implicated in the initiation, maintenance, and relapse of leukemia. The failure of current standard induction chemotherapy, which primarily targets dividing cells, has been linked to the quiescence of LSCs, leading to resistance to treatment. Consequently, there is an urgent need for novel therapies specifically aimed at eradicating LSCs to achieve more effective and durable treatment responses. CDK8 and its paralog CDK19 associate with the mediator complex and regulate gene expression programs linked to cancer and stem cell pluripotency. RVU120, a highly selective and potent CDK8/19 inhibitor, is currently undergoing clinical trials for AML and high-risk myelodysplastic syndrome (HR-MDS) (NCT04021368). Previous studies have demonstrated its potential mechanism of action on LSCs, wherein inhibition of CDK8/CDK19 led to the activation of lineage commitment genes in sensitive AML cells. Through recent gene expression analysis, it was discovered that inhibition of CDK8/CDK19 in transformed CD34+ cord blood cells resulted in the downregulation of stemness and quiescence-associated genes, while simultaneously upregulating genes associated with hematopoietic lineage-committed progenitors. Building on these findings, this study further investigates the potential of CDK8/CDK19 inhibition as a means to target LSCs, providing a rationale for RVU120 as a frontline treatment option in AML. The impact of RVU120 on LSCs has been investigated using an in vitro hierarchical AML-8227 model. This primary model maintains distinct populations in culture conditions, including CD34+CD38- cells with quiescent LSC potential, CD34+CD38+ cells enriched in progenitors, and CD34- more mature blasts (Lechman et al., 2016, Cancer Cell). The CD34+CD38- LSC population was further characterized by flow cytometry showing the expression of LSC surface markers, namely CD45RA, CD123, CD44, and negative expression for CD90, rendering it a suitable model for mechanistic studies. Next, AML-8227 cells were subjected to RVU120 treatment, resulting in a noticeable reduction in cell proliferation and viability. Flow cytometric analysis demonstrated a significant decrease in the percentage and total cell number of LSC-enriched/progenitor populations within the AML hierarchical model upon inhibition of CDK8/CDK19. Furthermore, RVU120 treatment induced the differentiation of cells, as evidenced by a sequential increase in the percentage of early myeloid differentiation marker, CD64, and late myeloid differentiation marker, CD14 (Fig. 1). In order to deconvolute the activity and mechanism of action of RVU120 in different cell populations, single-cell RNAseq analysis on AML-8227 cells has been performed at 24h and 3 days of treatment using technology from 10xGenomics. Additionally, analyzed AML-8227 cells have been enriched in the CD34+ population to evaluate the effect of RVU120 on stem and progenitor fraction at the single-cell level. The bioinformatic analysis of the performed experiment is ongoing and its results will be integrated into the data described above. CDK8 activity can be conveniently tracked by measuring specific phosphorylation of its target S726 on STAT5 protein. Dose and exposure-dependent inhibition of this marker has been established in cells exposed to RVU120. Levels of STAT5 S726 have been measured in different cell populations in patients from the first-in-human study of RVU120 in AML/HR-MDS. Consistent with established roles of CDK8 in LSCs, significantly elevated levels of pSTAT5 were observed in CD34+ stem/progenitor-enriched population when compared to CD34- blast cells measured at baseline before the start of the treatment (p&amp;lt;0.0001; ratio paired t-test; updated data from ASH 2022; Fig. 2). In summary, RVU120 displays cytotoxic and differentiating effects on well-characterized LSC populations in various models. Additionally, the CDK8 activity marker is directly associated with LSC-enriched bone marrow cells in AML patients, highlighting RVU120's potential as a promising therapeutic for targeting AML LSCs. Moreover, single-cell studies offer insights into its inhibitory effects on LSC-enriched populations and capacity for inducing differentiation. Overall, these results support RVU120 as a frontline candidate in AML therapy, countering therapeutic failure caused by persistent LSCs.

Molecular Mechanisms Underlying Abnormal Activation of T Cells in Idiopathic Aplastic Anemia and Hypoplastic Myelodysplastic Neoplasms

Idiopathic aplastic anemia (AA) and hypoplastic myelodysplastic neoplasms (MDS-h) are severe hematopoietic disorders characterized by pancytopenia and hypoplastic bone marrow. There is compelling evidence that these distinct clinical entities share a common pathophysiology based on the damage of hematopoietic stem and progenitor cells (HSPCs) by cytotoxic T cells. Expanded T cells overproduce proinflammatory cytokines, resulting in decreased proliferation and increased apoptosis of HSPCs. To uncover the molecular mechanisms underlying this abnormal immune response, we used RNA-Seq for the transcriptome analysis of T cells from patients with idiopathic AA and MDS-h. CD3+ cells were isolated from peripheral blood of 15 patients with AA or MDS-h at diagnosis and 6 healthy blood donors. The RNA-Seq library was constructed by NEB Next Ultra II Directional RNA Library Prep Kit and the data were analysed by R software 4.0.2. Functional annotation of differently expressed genes was performed using DAVID database and Gene Set Enrichment Analysis (GSEA). Principal component analysis-based clustering of RNA-Seq data defined the three major components for AA and MDS-h patients, and healthy individuals (Fig. 1A). As expected, the control samples formed a distinct group, meanwhile patient samples partially clustered together. The same results were obtained using unsupervised hierarchical clustering. The differential expression analysis (DEA) identified 256 significantly upregulated and 96 downregulated genes (ІlogFCІ&amp;gt;1, FDR&amp;lt;0.05) in patients compared to controls. Notably, the DEA detected many long noncoding RNAs (lncRNAs) including upregulated lncRNAs associated with immunological disturbances (e.g. CDC42-IT1 and NEAT1), and oncogenesis (e.g. ACAP2-IT1, and FAM238A). Particularly, NEAT1 positively regulates differentiation of CD4+ T cells into Th17 cells through STAT3 protein. Autoreactive T cells may be further stimulated by increased expression of S100A8/A9 detected in the patient cells. Gene Ontology (GO) analysis annotated the upregulated genes in AA/MDS-h to biological processes associated with oxygen transport, B cell receptor signaling pathway, innate immune response, positive regulation of inflammatory response, and apoptotic process, etc. (adjusted p&amp;lt;0.05) (Fig. 1B). The downregulated genes were significantly enriched in processes related to adaptive immune response, cell surface receptor signaling pathway, mitochondrial ATP synthesis coupled proton transport, etc. (adjusted p&amp;lt;0.05) (Fig.1B). B cell receptor signaling pathway was enriched by upregulated genes such as BANK1, MEF2C, and SYK whose deregulation is associated with autoimmune disorders. Herein, the activation of apoptosis is likely driven via AP-1 positive modulators that showed increased expressions in the patients. These modulators are essential for T cell activation and regulatory T cell differentiation. Pathway enrichment analysis determined the following signaling pathways (adjusted p&amp;lt;0.05): B cell receptor signaling pathway, osteoclast differentiation, oxidative phosphorylation, chemical carcinogenesis - reactive oxygen species, and cytokine-cytokine receptor interaction. The GSEA using the Hallmark gene sets showed that DNA Repair and Oxidative Phosphorylation were significantly negatively correlated with patient phenotype (Fig. 1C). Both GSEA and pathway analysis revealed that oxidative phosphorylation was supressed in patient-derived T cells. This is consistent with previous metabolic findings demonstrating that a transition from oxidative phosphorylation to glycolysis is a sign of T cell activation and it is critical for support of rapid cell growth. In conclusion, our study suggests the possible mechanisms of aberrant T cellular-immunity in idiopathic AA and MDS-h. Consistent with abnormal biological features of T cells, a large number of deregulated genes in the patients was involved in immune and inflammatory responses. Furthermore, our analyses revealed novel pathways, such as oxidative phosphorylation, as well as candidates ( TLR2, and CDC42-IT1) for functional studies. The deregulation of multiple lncRNAs in the patients indicates their implication in the molecular pathogenesis and thus their roles in bone marrow failure need to be explored. Supported by AZV (NU21-03-00565), and MH CZ-DRO (UHKT, 00023736).