STAT3 Binding Research Articles

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

STAT3 and SOX-5 induce BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells

Retinoid-related orphan receptor gamma t (RORγt) is the lineage-specific transcription factor for T helper 17 (Th17) cells. Our previous study demonstrated that STAT3 likely participates in the activation of RORCE2 (a novel enhancer of the RORγt gene) in Th17 cells. However, the detailed mechanism is still unclear. Here, we demonstrate that both STAT3 and SOX-5 mediate the enhancer activity of RORCE2 in vitro. Deletion of the STAT3 binding site (STAT3-BS) in RORCE2 impaired RORγt expression and Th17 differentiation, resulting in reduced severity of experimental autoimmune encephalomyelitis (EAE). Mechanistically, STAT3 and SOX-5 bind the RORCE2 region and recruit the chromatin remodeling factor BRG1 to remodel the nucleosomes positioned at this region. Collectively, our data suggest that STAT3 and SOX-5 mediate the differentiation of Th17 cells through the induction of BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells.

Therapeutic Targeting of JAK/STAT Signaling and Histone Acetyltransferase Activity in Post-Myeloproliferative Neoplasm Secondary AML

Post-myeloproliferative neoplasm secondary Acute Myeloid Leukemia (Post-MPN sAML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms (MPN). Hyperactivation of JAK/STAT signaling is present in 50-90% of MPNs, underscoring its role in driving MPN pathogenesis and transformation to acute sAML. Ruxolitinib, a Type-I JAK2 inhibitor approved for the treatment of myelofibrosis, was tested in a Phase II clinical trial in post-MPN sAML. Despite enhancing patients' quality of life, ruxolitinib did not exert major improvements on disease outcome (Eghtedar et al, 2012). Thus, there is an urgent need to find synergistic drug combinations capable of complementing the therapeutic activity of JAK2 inhibitors in post-MPN sAML. Here, we demonstrate that combination treatment of ruxolitinib and CBP30, a bromodomain inhibitor of histone acetyltransferases (HAT) CREBBP and p300, enhances the therapeutic activity of ruxolitinib in post-MPN sAML using high throughput genetic screens, in vitro functional assays, transcriptomic and epigenetic profiling and mouse xenograft in vivo experiments. To identify druggable determinants of JAK2 inhibitor response in post-MPN sAML, we performed genome-wide loss-of-function CRISPR screens in sAML HEL cells treated with four different JAK2 inhibitors, including ruxolitinib. We discovered that depletion of the histone acetyl transferase CREBBP sensitizes HEL cells to JAK2 inhibition (Figure 1A). Pharmacological inhibition of JAK/STAT signaling and HAT activity using a combination of ruxolitinib and CREBBP inhibitors significantly reduced the proliferation of sAML cells HEL and SET2 (Figure 1B). Among the CREBBP inhibitors, CBP30 synergizes best with ruxolitinib having Chou-Talalay combination indices of 0.152 and 0.704 in HEL and SET2, respectively. Moreover, combination treatment of ruxolitinib and CBP30 markedly increased apoptosis and induced cell cycle arrest at G1 in HEL. To mechanistically characterize the synergism between ruxolitinib and CBP30, we performed ChIP-seq to profile STAT5 binding and H3K27ac levels in HEL cells treated with DMSO, ruxolitinib, CBP30 and their combination. STAT5 binding profiles did not reveal major differences in ruxolitinib versus ruxolitinib + CBP30 treated cells, suggesting that the drug combination does not significantly alter STAT5 binding. Conversely, we noticed striking global changes in H3K27ac levels in the combination compared with DMSO or single agents, indicating that the synergistic effect between ruxolitinib and CBP30 is mediated by substantial changes in transcriptional regulation. Notably, analysis of H3K27ac downregulated and upregulated sites identified enrichments in motifs recognized by FOS/SMAD4 and GATA transcription factors, respectively. These TFs are involved in the regulation of myeloid differentiation implicating a synergistic regulation of relevant myeloid differentiation pathways by the combination of JAK2 and CREBBP inhibitors in sAML. Furthermore, ruxolitinib + CBP30 treatment in HEL elicits a unique transcriptome profile with expression deregulation of genes associated with cell cycle, DNA repair, HAT activity and TGF-beta signaling as determined by DAVID functional annotation analysis. Lastly, to evaluate the synergistic effects of ruxolitinib and CBP30 in vivo, we treated luciferase-expressing HEL sAML mouse xenografts with vehicle, ruxolitinib (80 mg/kg), CBP30 (30 mg/kg) and ruxolitinib (80 mg/kg) + CBP30 (30 mg/kg). Combination of ruxolitinib and CBP30 displayed significant decrease in leukemia burden after 4 weeks of treatment as monitored by bioluminescence imaging and quantification. Consistently, mice treated with both drugs also showed remarkable decrease in their white blood cell count and spleen size compared with single-agent-treated and vehicle-treated mice. Collectively, our results substantiate the combinatorial therapeutic targeting of JAK/STAT signaling and HAT activity as a potential treatment approach for post-MPN sAML.

Distinct Role of STAT3 and STAT5 Factors in MDS and AML

Introduction: Myelodysplastic Syndrome (MDS) refers to a heterogeneous group of clonal hematologic disorders characterized by hematopoietic dysfunctions and impaired differentiation in the hematopoietic stem and progenitor cells compartment. MDS is commonly referred to as a pre-leukemic stage due to the increased risk of progression to Acute Myeloid Leukemia (AML). AML is an aggressive hematologic malignancy characterized by the accumulation of immature myeloblasts in the bone marrow and the peripheral blood. Signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) are important regulators of several cellular processes including cell proliferation and survival. Abnormal STAT3 and STAT5 signaling has been implicated in various solid and hematologic malignancies, including MDS and AML, which renders them appealing targets for the development of novel therapeutic strategies. Materials: STAT3, STAT5A or STAT5B knock-downs have been generated in two AML (MOLM-13 and Kasumi-1) and two MDS (MDS92 and MDS-L, gift of Prof. Kaoru Tohyama) cell lines, using shRNAs through lenti-viral delivery. The 4 biological replicates with the highest levels of knock-downs for STAT5A, STAT5B or STAT3 have been selected for each cell line and mRNA sequencing has been performed. To define the genome-wide binding of STAT5A, STAT5B and STAT3 in the two conditions, CUT&Tag has been performed in wild type MOLM-13, Kasumi-1 and MDS-L cells. Additionally, ATAC sequencing has been carried out in the same cell lines to investigate the chromatin conformation changes between the two conditions. Furthermore, STAT3, STAT5A and STAT5B knock-downs have been conducted using shRNAs through lenti-viral delivery in CD34+ enriched cells, isolated from bone marrow mononuclear cells of 14 high-risk MDS patients and 14 secondary (MDS-related) AML patients. The samples have been processed for mRNA-Sequencing to model the leukemic transformation in primary human cells. Results: To elucidate the role of STAT3 and STAT5 in MDS to AML transformation, changes in STAT3 and STAT5 target gene networks have been identified. mRNA sequencing data from STAT3, STAT5A or STAT5B knock-downs in MDS and AML cell lines revealed a distinct role of the three factors in each state. The differential binding of each factor, explored through CUT&Tag, and the gene expression profiles in MDS and AML reciprocally reveal a different function of each factor, including pathways involved in cell cycle and apoptosis. Also, the direct and indirect target genes of STAT3, STAT5A and STAT5B in each condition, as well as a possible interplay between STAT3 and STAT5 activation in AML has been uncovered. The exploration of the chromatin accessibility landscapes between the MDS and AML conditions through ATAC sequencing has provided an insight into the chromatin conformation states, unveiling potential leukemia promoting networks, as well as the involvement of STAT3 and STAT5 pathways in the malignant transformation. Furthermore, a potential cross talk of STAT3 and STAT5 factors with the IKAROS family members, has been linked to AML transformation, as significant differential regulation has been detected between MDS and AML conditions after knock-down of STAT5A and STAT5B. The results are currently validated in primary hematopoietic cells of MDS and AML patients after STAT3, STAT5A or STAT5B knock-downs. Conclusions: Our results show a different role between the two STAT5 factors (STAT5A and STAT5B) in MDS and AML conditions and delineate the role of STAT5A, STAT5B and STAT3 factors and their target gene networks in leukemic transformation, thus providing novel targets for therapeutic management of MSD and AML. Funding: This project is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 (ARCH)

Molecular Profiling of Response and Resistance to Venetoclax-Decitabine Therapy in Acute Myeloid Leukemia

The hypomethylating agent decitabine and the BCL-2 inhibitor venetoclax combination (DEC-VEN) is now standard of care in AML, yet a subset of patients remains refractory, and factors that reduce efficacy remain to be fully elucidated. This study aimed to systematically identify molecular determinants of resistance via single-cell (sc)RNA-seq and scATAC-seq analysis of bone marrow (BM) samples from patients participating in a prospective clinical trial of DEC-VEN (NCT03404193). For scRNA-seq, 17 BM samples (total 39,607 cells) from 12 AML patients (median age 67; 8 responders; 4 non-responders; 5 pre- and post-treatment pairs) and 3 healthy BM controls were integrated and annotated into 23 transcriptional cell clusters. In the Uniform Manifold Approximation and Projection (UMAP) plot, C1 overlapped with healthy hematopoietic stem cells (HSCs), C2 appeared only in AML patients, and C3 overlapped with progenitor cells. C1 was characteristic of refractory AML and persisted after DEC-VEN treatment. (Figure 1) C1 cells showed significantly higher expression of FLT3, BCL2, MCL1 and transcription factors MYB, ETV6, RUNX1, LMO2, JUND compared to HSCs from healthy donors. DEC-VEN treatment markedly increased ribosomal protein ( RP ) genes and mitochondrial electron transfer COX family genes and increased expression of the leukemia stem cell (LSC) marker CD74 in C1 of non-responders. In C2 cells, high expression of LSC marker genes FLT3, PIM1, MYC and transforming master regulator genes MYB, ETV6, RUNX2, LMO2, STAT3, NOTCH1, BCL11A was observed. DEC-VEN significantly upregulated RP genes and CD74 in C2 in non-responders, as in C1. In responders, hemoglobin family genes were significantly increased in C2. C3 cells were characterized by highly expressed iron metabolism genes, which were further increased by DEC-VEN in responders. GSEA analysis demonstrated that DEC-VEN upregulated ATP metabolic processes and ribosomal biosynthesis and downregulated stem cell differentiation genes in refractory C1 and C2. In responders' AML clusters, DEC-VEN upregulated cellular oxidant detoxification and iron ion homeostasis and downregulated myeloid leukocyte activation. Next, we extended the AML transcriptome analysis by profiling accessible chromatin by scATAC-seq using BM from pre- and post- DEC -VEN treatment of 2 AML patients (1 refractory, 1 relapsed after remission with incomplete count recovery [CRi]), and 2 healthy controls (total 41,062 cells). Leukemia-like cluster (LLC) cells showed significantly higher chromatin accessibility of CD74, RUNX1, RUNX2, NOTCH1, LMO2, ETV6, JUND, STAT3, BCL2, BCL11A and enriched binding motifs of RUNX2, ETV6, MYB, JUN, JUND, STAT3 compared to other clusters, which was consistent with gene expression findings of C1 and C2 by scRNA-seq. DEC-VEN treatment induced further increased motif enrichment of STAT3 in LLC in CRi AML, and STAT3 RUNX2, IKZF1 in refractory AML. To interpret T cell kinetics in response to DEC-VEN in AML, T cells from scRNA-seq profiling were subclassified based on canonical gene expression. The results showed that the CD3+ and CD3+CD8+ cell signatures were significantly lower in non-responders compared to responders post-treatment (p<0.005). Notably, the ratio of exhausted CD8+ cell clusters was significantly higher in non-responders than in responders in DEC-VEN post-treatment (p<0.05), with downregulation of CD8+ effector marker GZMK, and regulator of CD8+ cytotoxic T cell development TOX. In summary, we identified a subpopulation of AML cells that overlap with HSCs in UMAP plots and are associated with DEC-VEN resistance. LSC-related genes were enriched in resistant AML clusters, while hemoglobin-related genes were increased in sensitive clusters. DEC-VEN induced mitochondrial metabolism genes in resistant cells and further enhanced erythrocytic differentiation in sensitive cells. In addition, scATAC-seq revealed LSC-specific chromatin accessibility patterns, and depletion of CD8+ T cells was observed in non-responders. These findings shed light on the molecular mechanisms of DEC-VEN resistance.

LRH-1 agonist DLPC through STAT6 promotes macrophage polarization and prevents parenteral nutrition-associated cholestasis in mice.

Parenteral nutrition-associated cholestasis (PNAC) is an important complication in patients with intestinal failure with reduced LRH-1 expression. Here, we hypothesized that LRH-1 activation by its agonist, dilauroylphosphatidylcholine (DLPC), would trigger signal transducer and activator of transcription 6 (STAT6) signaling and hepatic macrophage polarization that would mediate hepatic protection in PNAC. PNAC mouse model (oral DSSx4d followed by PNx14d; DSS-PN) was treated with LRH-1 agonist DLPC (30mg/kg/day) intravenously. DLPC treatment prevented liver injury and cholestasis while inducing hepatic mRNA expression of Nr5a2 (nuclear receptor subfamily 5 group A member 2), Abcb11 (ATP binding cassette subfamily B member 11), Abcg5 (ATP-binding cassette [ABC] transporters subfamily G member 5), Abcg8 (ATP-binding cassette [ABC] transporters subfamily G member 8), nuclear receptor subfamily 0, and ATP-binding cassette subfamily C member 2 ( Abcc2) mRNA, all of which were reduced in PNAC mice. To determine the mechanism of the DLPC effect, we performed RNA-sequencing analysis of the liver from Chow, DSS-PN, and DSS-PN/DLPC mice, which revealed DLPC upregulation of the anti-inflammatory STAT6 pathway. In intrahepatic mononuclear cells or bone-marrow derived macrophages (BMDM) from PNAC mice, DLPC treatment prevented upregulation of pro-inflammatory (M1) genes, suppressed activation of NFκB and induced phosphorylation of STAT6 and its target genes, indicating M2 macrophage polarization. In vitro, incubation of DLPC with cultured macrophages showed that the increased Il-1b and Tnf induced by exposure to lipopolysaccharides or phytosterols was reduced significantly, which was associated with increased STAT6 binding to promoters of its target genes. Suppression of STAT6 expression by siRNA in THP-1 cells exposed to lipopolysaccharides, phytosterols, or both resulted in enhanced elevation of IL-1B mRNA expression. Furthermore, the protective effect of DLPC in THP-1 cells was abrogated by STAT6 siRNA. These results indicate that activation of LRH-1 by DLPC may protect from PNAC liver injury through STAT6-mediated macrophage polarization.

Cutting Edge: Bach2 Integrates Cytokine Signals to Arbitrate Differentiation Decisions between T Follicular Helper and Th17 Lineages.

CXCR5 is a hallmark of T follicular helper (Tfh) cells. The mechanism of CXCR5 induction, however, is still incompletely understood. In this study, we report that in mice with the absence of transcription factor Bach2, the Th17-inducing cytokines IL-6 and TGF-β together induced CXCR5 expression invitro. Mechanistically, IL-6/STAT3 drove Cxcr5 promoter activity via the upstream site 1 regulatory element, whereas TGF-β enhanced permissive histone modifications, and the STAT3 binding to the site 1 regulatory element was higher in the absence of Bach2. Subsequently, despite previous studies showing enhanced Th17 cell differentiation in the absence of Bach2 invitro, we found that invivo, the Bach2 deficiency led to an enhanced Tfh cell response at the expense of the Th17 cell response. These findings suggest that Bach2 helps integrate cytokine signals to arbitrate differentiation decisions between Tfh and Th17 lineages.

Cabozantinib inhibits HBV-RNA transcription by decreasing STAT3 binding to the enhancer region of cccDNA.

Precision medicine and customized therapeutics based on the features of each patient are important for maximizing therapeutic effects. Because most cases of HCC occur in the damaged liver through various etiologies, such as hepatitis virus infection, steatohepatitis, and autoimmune hepatitis, there should be a rationale for the choice of therapeutic options based on these etiologies. Although cabozantinib, an oral multikinase inhibitor, has demonstrated clinical effectiveness in advanced HCC, subgroup analyses showed a lower HR for death in HBV-related HCC. This study aimed to determine the therapeutic effects of cabozantinib in HBV-related HCC. Using HBV infection models and gene knockout cells, we determined the crucial signaling axis responsible for the effects of cabozantinib on HBV. A chromatin immunoprecipitation assay was performed to determine the interaction between the signaling molecules and HBV DNA. Agonists and inhibitors were used for confirmation. Cabozantinib inhibited HBV replication through the HGF-mesenchymal-epithelial transition factor-signal transducer and activator of transcription 3 (MET-STAT3) signaling axis. The importance of STAT3 in viral replication has been confirmed using gene-edited STAT3 knockout cells. The chromatin immunoprecipitation assay revealed that the binding levels of phosphorylated STAT3 to enhancer region 1 of HBV covalently closed circular DNA were significantly increased by HGF stimulation. Cabozantinib has favorable therapeutic effects on HBV-related HCC because it inhibits HCC not only directly but also indirectly by means of inhibitory effects on HBV.

TTC13 expression and STAT3 activation may form a positive feedback loop to promote ccRCC progression

Background Renal cell carcinoma (RCC) originates from renal tubular epithelial cells and is mainly classified into three histological types, including clear cell renal cell carcinoma (ccRCC) which accounts for about 75% of all kidney cancers and is characterized by its strong invasiveness and poor prognosis. Hence, it is imperative to understand the mechanisms underlying the occurrence and progression of ccRCC to identify effective biomarkers for the early diagnosis and the prognosis prediction. Methods The mRNA level of TTC13 was quantified by RT-PCR, while the protein level was determined by western blot and immunohistochemistry (IHC) staining. Cell proliferation was measured by cck-8, and cell apoptosis was detected by flow cytometry. The binding of STAT3 to the promoter region of TTC13 was determined by the luciferase reporter assay and chip experiments. STAT3 nuclear translocation was assessed by immunofluorescence staining. Results We found that TTC13 was up-regulated in ccRCC, and TTC13 promoted cell proliferation as well as inhibited cell apoptosis and autophagy of ccRCC through wnt/β-catenin and IL6-JAK-STAT3 signaling pathways. Furthermore, TTC13 might play a role in the immune infiltration and immunotherapy of ccRCC. Mechanistically, STAT3 activated the transcription of TTC13 gene. Conclusions STAT3 directly regulated TTC13 expression through a positive feedback loop mechanism to promote ccRCC cell proliferation as well as reduce cell apoptosis and autophagy. These findings suggested new and effective therapeutic targets for more accurate and personalized treatment strategies.

Identification the immune related marker genes and transcription-factor network in ruptured cerebral aneurysms using bioinformatics analysis and machine-learning strategies

BackgroundAs a high level of mortality and morbidity disease, ruptured cerebral aneurysms (RCA) is the most common cause of intracranial hemorrhage. MethodsThe expression profiles of GSE13353, GSE26969 were downloaded from the Gene Expression Omnibus (GEO) database to obtain differentially expressed genes (DEGs) between RCA and unruptured cerebral aneurysms (URCA). The Least Absolute Shrinkage Selection Operator (LASSO) and the SVM-RFE (SVM-RFE) analysis were used to screen the potential diagnostic genes for RCA, which were further tested in the validation cohort (GSE54083). The TRRUST database was used to predict transcription factors (TF) and the JASPAR database was used to validate the marker genes and STAT1 binding sites. Finally, the CMAP database was used to predict RCA-associated drugs for clinical transformation. ResultsA total of 120 DEGs were identified. The functional enrichment analysis revealed that NF-kappa B signaling pathway, tuberculosis, rheumatoid arthritis were enriched in the RCA group. NANS, NTRK3, OLFML2A were identified as diagnostic biomarkers of RCA. Transcription of all three marker genes is regulated by the predicted transcription factor STAT1, the CMAP database was eventually used to screen 10 most promising drugs for the treatment of RCA. ConclusionNANS, NTRK3, OLFML2A may become new candidate biomarkers of RCA, moreover, STAT1 plays an important role in the development of RCA. The mentioned immune cells may play a key role in the development and progression of RCA. MEK and MAP kinase inhibitors are the most likely drug mechanisms for RCA therapy.

AXL transcriptionally up-regulates TMEM14A expression to mediate cell proliferation in non-small-cell lung cancer cells

In non-small cell lung cancer (NSCLC), the receptor tyrosine kinase AXL has been identified as a potent activator of tumor progression and resistance to therapies. However, the molecular mechanisms behind AXL-mediated oncogenesis remain elusive. Current study thus aimed to uncover potential downstream genes regulated by AXL in NSCLC. Through transcriptomic RNA sequencing of AXL-silenced NSCLC cells, TMEM14A was identified as a significantly up-regulated gene. Clinical evaluations using GEPIA2 revealed that TMEM14A mRNA expression was notably higher in lung adenocarcinoma (LUAD) tumor tissues compared to normal tissues. Further, significantly increased TMEM14A levels were associated with poorer overall survival in LUAD patients. Experimentally, silencing TMEM14A in NSCLC cells led to reduced cellular proliferation and ATP levels, highlighting a key role of TMEM14A in NSCLC progression. Moreover, our promoter analysis demonstrated that AXL-mediated regulation of TMEM14A transcription could involve binding of transcription factors STAT and NF-κB to 5′-promoter of TMEM14A. Collectively, current study unveils TMEM14A as a novel downstream target of AXL, suggesting its potential as a therapeutic target to counteract resistance in future NSCLC patients undergoing AXL-targeted therapies.

KLF12 transcriptionally regulates PD-L1 expression in non-small cell lung cancer.

Recent studies have pointed to the role of Krüpple-like factor 12 (KLF12) in cancer-associated processes, including cancer proliferation, apoptosis, and metastasis. However, the role of KLF12 in tumor immunity remains obscure. Here, we found that KLF12 expression was significantly higher in non-small cell lung cancer (NSCLC) cells with higher programmed death-ligand 1 (PD-L1) expression. Additionally, a positive correlation between KLF12 and PD-L1 was observed in clinical patient tumor tissues. By chromatin immunoprecipitation (ChIP) analysis, KLF12 was identified to bind to the CACCC motif of the PD-L1 promoter. Overexpression of KLF12 promoted PD-L1 transcription, whereas silencing of KLF12 inhibited PD-L1 transcription. Furthermore, signal transducer and activator of transcription 1 (STAT1)- and STAT3-triggered PD-L1 transcription was abolished in the absence of KLF12, and KLF12 knockdown weakened the binding of STAT1 and STAT3 to the PD-L1 promoter. Mechanistically, KLF12 physically interacted with P300, a histone acetyltransferase. In addition, KLF12 silencing reduced P300 binding to the PD-L1 promoter, which subsequently caused decreased acetylation of histone H3. PD-L1 transcription driven by KLF12 overexpression was eliminated by EP300 silencing. In immunocompetent mice, KLF12 knockout inhibited tumor growth and promoted infiltration of CD8+ T cells. However, this phenomenon was not observed in immunodeficient mice. Overall, this study reveals KLF12-mediated transcriptional regulation of PD-L1 in NSCLC; targeting KLF12 may be a potential therapeutic strategy for NSCLC.

Sanguinarine induces apoptosis in osteosarcoma by attenuating the binding of STAT3 to the single-stranded DNA-binding protein 1 (SSBP1) promoter region.

Osteosarcoma, a primary malignant bone tumour prevalent among adolescents and young adults, remains a considerable challenge despite protracted progress made in enhancing patient survival rates over the last 40 years. Consequently, the development of novel therapeutic approaches for osteosarcoma is imperative. Sanguinarine (SNG), a compound with demonstrated potent anticancer properties against various malignancies, presents a promising avenue for exploration. Nevertheless, the intricate molecular mechanisms underpinning SNG's actions in osteosarcoma remain elusive, necessitating further elucidation. Single-stranded DNA-binding protein 1 (SSBP1) was screened out by differential proteomic analysis. Apoptosis, cell cycle, reactive oxygen species (ROS) and mitochondrial changes were assessed via flow cytometry. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) were used to determine protein and gene levels. The antitumour mechanism of SNG was explored at a molecular level using chromatin immunoprecipitation (ChIP) and dual luciferase reporter plasmids. Our investigation revealed that SNG exerted an up-regulated effect on SSBP1, disrupting mitochondrial function and inducing apoptosis. In-depth analysis uncovered a mechanism whereby SNG hindered the JAK/signal transducer and activator of transcription 3 (STAT3) signalling pathway, relieved the inhibitory effect of STAT3 on SSBP1 transcription, and inhibited the downstream PI3K/Akt/mTOR signalling axis, ultimately activating apoptosis. The study delved further into elucidating the anticancer mechanism of SNG in osteosarcoma. Notably, we unravelled the previously undisclosed apoptotic potential of SSBP1 in osteosarcoma cells. This finding holds substantial promise in advancing the development of novel anticancer drugs and identification of therapeutic targets.

The CSN5/HSF/SPI1/PU.1 Axis Regulates Cell Proliferation in Hypocellular Myelodysplastic Syndrome Patients.

This study explored the relationship between the activation of the jak/stat3 signaling pathway and the CSN5 gene transcript and protein expression levels in the hematopoietic stem cells of patients with myelodysplastic syndromes (MDSs). This study also aimed to investigate the correlation between the expression level of CSN5 and the deubiquitination of HSF1, as well as the transcript level of the spi1/pu.1 genes to explore the pathogenesis of MDS. We isolated cells from normal individuals and MDS patients, and the mRNA and protein expression levels of spi1/pu.1 in cd34+ cells (hematopoietic stem cells) were measured by PCR and western blotting, respectively. A ChIP assay was used to detect the binding of HSF1 to the spi1/pu.1 promoter in cd34+ cells. The ubiquitination of HSF1 in cd34+ cells was detected by CO-IP. The binding of HSF1 and Fbxw7α was detected in in cd34+ cells by CO-IP. The binding of HSF1 and CSN5 was evaluated. A luciferase reporter assay was used to detect the effect of STAT3 on CSN5 promoter activation in cd34+ cells. Western blotting was used to detect the phosphorylation of STAT3 in cd34+ cells of MDS patients. The binding of STAT3 and C/EBP beta in cd34+ cells was detected by CO-IP. Inhibition of SPI1/PU.1 expression was observed in MDS samples with low proliferation ability. Further experiments proved that phosphorylation of STAT3 affected CSN5 function and mediated the ubiquitination of HSF, the upstream regulator of SPI1/PU.1 transcription, which led to the inhibition of SPI1/PU.1 expression. Restoration of CSN5 rescued the inhibition of HSF1 ubiquitination, causing SPI1/PU.1 transcription to resume and increasing SPI1/PU.1 expression, promoting the recovery of cell proliferation in hypocellular MDS. Our research revealed the regulatory role of the CSN5/HSF/SPI1/PU.1 axis in hypocellular MDS, providing a probable target for clinical intervention.

ROR1/STAT3 positive feedback loop facilitates cartilage degeneration in Osteoarthritis through activation of NF-κB signaling pathway.

Osteoarthritis (OA) is a chronic joint disorder with a serious impact on society. The main pathological change in OA is articular cartilage degeneration, which is directly associated with imbalance of anabolic and catabolic activities in chondrocytes. To evaluate the expression and biological effects of ROR1 in OA cartilage and determine whether knockdown of ROR1 attenuates cartilage degeneration. ROR1 expression in OA clinical specimens was evaluated by western blotting and immunohistochemistry. The effects of ROR1 on anabolic and catabolic activities were evaluated in Wnt5a-treated human primary chondrocytes by western blotting, immunofluorescence, and luciferase assay. The effects of ROR1 knockdown on cartilage degeneration in a surgical OA mouse model were examined by X-ray imaging and Safranin O-Fast Green histological staining. ROR1 was considerably upregulated in cartilage tissues of OA patients. ROR1 knockdown alleviated the activation of the NF-κB signaling pathway and reversed the suppression of collagen II and aggrecan by Wnt5a, as well as upregulation of ADAMTS-5 and MMP-13 in chondrocytes. In addition, ROR1 knockdown significantly reduced Wnt5a-induced STAT3 nuclear translocation. STAT3 binding to the ROR1 promoter indicated a positive feedback loop between ROR1 and STAT3. ROR1 knockdown was confirmed to dramatically alleviate cartilage degradation in the DMM induced-OA mouse model. Increased expression of ROR1 in OA cartilage tissues leads to a positive feedback loop with STAT3, which activates the NF-κB signaling pathway, resulting in an imbalance between chondrocyte anabolism and catabolism. These results indicate a potential new therapeutic target for the treatment of OA.

Single-cell multi-omics sequencing reveals the immunological disturbance underlying STAT3-V637M Hyper-IgE syndrome

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by, among others, the excessive production of IgE and repetitive bacterial/fungal infections. Mutations in STAT3, a transcription factor that orchestrates immune responses, may cause HIES, but the underlying mechanisms are not fully understood. Here, we used multi-omic approaches to comprehensively decipher the immune disturbance in a male HIES patient harboring STAT3-V637M. In his peripheral blood mononuclear cell (PBMC) we found significant clonal expansion of CD8 T cells (with increased CD8 subunits expression, potentially enhancing responsiveness to MHC I molecules), but not in his CD4 T cells and B cells. Although his B cells exhibited a higher potential in producing immunoglobulin, elevated SPIC binding might bias the products toward IgE isotype. Immune checkpoint inhibitors, including CTLA4, LAG3, were overexpressed in his PBMC-CD4 T cells, accompanied by reduced CD28 and IL6ST (gp130) expression. In his CD4 T cells, integrative analyses predicted upstream transcription factors (including ETV6, KLF13, and RORA) for LAG3, IL6ST, and CD28, respectively. The down-regulation of phagocytosis and nitric oxide synthesis-related genes in his PBMC-monocytes seem to be the culprit of his disseminated bacterial/fungal infection. Counterintuitively, in his PBMC we predicted increased STAT3 binding in both naïve and mature CD4 compartments, although this was not observed in most of his PBMC. In his bronchoalveolar lavage fluid (BALF), we found two macrophage subtypes with anti-bacterial properties, which were identified by CXCL8/S100A8/S100A9, or SOD2, respectively. Together, we described how the immune cell landscape was disturbed in STAT3-V637M HIES, providing a resource for further studies.

Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis.

Clonal hematopoiesis (CH) increases the risk of atherosclerotic cardiovascular disease possibly due to increased plaque inflammation. Human studies suggest that limitation of interleukin-6 (IL-6) signaling could be beneficial in people with large CH clones, particularly in TET2 CH. Here we show that IL-6 receptor antibody treatment reverses the atherosclerosis promoted by Tet2 CH, with reduction of monocytosis, lesional macrophage burden and macrophage colony-stimulating factor 1 receptor (CSF1R) expression. IL-6 induces expression of Csf1r in Tet2-deficient macrophages through enhanced STAT3 binding to its promoter. In mouse and human Tet2-deficient macrophages, IL-6 increases CSF1R expression and enhances macrophage survival. Treatment with the CSF1R inhibitor PLX3397 reversed accelerated atherosclerosis in Tet2 CH mice. Our study demonstrates the causality of IL-6 signaling in Tet2 CH accelerated atherosclerosis, identifies IL-6-induced CSF1R expression as a critical mechanism and supports blockade of IL-6 signaling as a potential therapy for CH-driven cardiovascular disease.

STAT1 is required to establish but not maintain interferon‐γ‐induced transcriptional memory

Exposure of human cells to interferon‐γ (IFNγ) results in a mitotically heritable yet reversible state called long‐term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFNγ. Here, we discovered that in primed cells, both interferon‐responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re‐exposure to IFNγ, specifically at promotors of primed genes. Priming does not alter the degree of IFNγ‐induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK–STAT signaling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that the memory of interferon exposure constitutes a STAT1‐mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

Th1 Cells Alter the Inflammatory Signature of IL-6 by Channeling STAT Transcription Factors to Alu-like Retroelements.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

STA-21, a small molecule STAT3 inhibitor, ameliorates experimental autoimmune encephalomyelitis by altering Th-17/Treg balance.

Numerous studies have demonstrated the role of T helper (Th) 17 and T regulatory (reg) cells and pro-inflammatory and anti-inflammatory cytokines related to these cells in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). STAT3 is one of the downstream signaling proteins of IL-23, IL-6, and IL-21 that are required for Th17 cells differentiation. STA-21 is a STAT3 inhibitor that functions by inhibiting STAT3 dimerization and binding to DNA impairing the expression of STAT3 target genes including, RORγt, IL-21 and IL-23R that are also required for Th17 cell differentiation. In this study, we evaluated the effect of STA-21 on EAE Model and investigated how this small molecule can change Th17/Treg balance leading to amelioration of disease. After EAE induction and treatment with STA-21, its effects were assessed. Major assays were H&E and LFB staining, Flow cytometric analysis, Reverse transcription-PCR (RT-PCR), and ELISA. STA-21 ameliorated the EAE severity and decreased the EAE inflammation and demyelination. It also decreased STAT3 phosphorylation, the proportion of Th17 cells and the protein level of IL-17. In contrast, the balance of Tregs and the level of anti-inflammatory cytokine, IL-10 increased in STA-21-treated mice. Moreover, STA-21 significantly decreased the expression of Th17 related transcription factors, RORɣt and IL-23R while FOXP3 expression associated with Treg differentiation was increased. This study showed that STA-21 has therapeutic effects in EAE by reducing inflammation and shifting inflammatory immune responses to anti-inflammatory and can be used as a suitable treatment strategy for the treatment of EAE. The effectiveness of inhibiting or strengthening the functional cells of the immune system by these small molecules in terms of easy to access, simple construction and inexpensive expansion make them as a suitable tool for the treatment of inflammatory and autoimmune diseases.