Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.

Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.

Plakevulin A induces apoptosis and suppresses IL-6-induced STAT3 activation in HL60 cells.

(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and ß, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-ß dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.

Research progress of multi-target HDAC inhibitors blocking the BRD4-LIFR-JAK1-STAT3 signaling pathway in the treatment of cancer.

Histone deacetylase inhibitors (HDACis) show beneficial effects on different hematological malignancy subtypes. However, their impacts on treating solid tumors are still limited due to diverse resistance mechanisms. Recent studies have found that the feedback activation of BRD4-LIFR-JAK1-STAT3 pathway after HDACi incubation is a vital mechanism inducing resistance of specific solid tumor cells to HDACis. This review summarizes the recent development of multi-target HDACis that can concurrently block BRD4-LIFR-JAK1-STAT3 pathway. Moreover, our findings hope to shed novel lights on developing novel multi-target HDACis with reduced BRD4-LIFR-JAK1-STAT3-mediated drug resistance in some tumors.

Development of new thieno[2,3-d]pyrimidines as dual EGFR and STAT3 inhibitors endowed with anticancer and pro-apoptotic activities.

In part due to the resilience of cellular feedback pathways that develop therapeutic resistance to targeting the EGFR alone, using EGFR inhibitors alone was demonstrated to be unsuccessful in clinical trials. The over-activation of the signal transducer/activator of transcription 3 (STAT3) during the administration of an EGFR inhibitor is expected to play a substantial part in the failure and resistance of EGFR inhibitor treatment. Therein, we proposed a hypothesis that induced STAT3-mediated resistance to EGFR inhibition therapy could be addressed by a dual inhibition of EGFR and STAT3 method. To this end, we tried to discover new thieno[2,3-d]pyrimidine derivatives "5a-o". Results from the screening on A549 and MCF7 cancer cell lines revealed that compounds 5j and 5k showed two-digit nanomolar with appropriate safety towards the WI-38 cell line. The best molecules, 5j and 5k, were subjected to γ-radiation, and their cytotoxic efficacy didn't change after irradiation, demonstrating that not having to use it avoided its side effects. Compounds 5j and 5k demonstrated the highest inhibition when their potency was tested as dual inhibitors on EGFR 67 and 41 nM, respectively, and STAT3 5.52 and 3.34 nM, respectively, proved with in silico molecular docking and dynamic simulation. In light of the results presented above, the capacity of both powerful compounds to alter the cell cycle and initiate the apoptotic process in breast cancer MCF7 cells was investigated. Caspase-8, Bcl-2, Bax and Caspase-9 apoptotic indicators were studied.

Targeting JAK2/STAT3 for the treatment of cancer: A review on recent advancements in molecular development using structural analysis and SAR investigations.

Cancer is indeed considered a hazardous and potentially life-threatening disorder. The JAK/STAT pathway is an important intracellular signaling cascade essential for many physiological functions, such as immune response, cell proliferation, and differentiation. Dysregulation of this pathway aids in the progression and development of cancer. The downstream JAK2/STAT3 signaling cascades are legitimate targets against which newer anticancer drugs can be developed to prevent and treat cancer. Understanding the mechanisms behind JAK2/STAT3 participation in cancer has paved the way for developing innovative targeted medicines with the potential to improve cancer treatment outcomes. This article provides information on the current scenario and recent advancements in the design and development of anticancer drugs targeting JAK2/STAT3, including structural analysis and SAR investigations of synthesized molecules. Numerous preclinical and clinical trials are ongoing on these inhibitors, which are highlighted to gain more insight into the broader development prospects of inhibitors of JAK2/STAT3.

Inhibition of the STAT3-EPHX2 axis promotes regression of ulcerative colitis by treatment with novel porphyrin derivative.

LD4, a novel porphyrin derivative, has attracted much attention for its excellent anti-inflammatory properties. It can promote the healing of colonic mucosa, reduce inflammatory response, regulate oxidative stress, and thus improve ulcerative colitis (UC) symptoms. However, the specific signaling pathways of LD4-PDT involved in UC have not been explored. The present study aimed to elucidate the effects of LD4 on UC and to investigate the underlying mechanisms both in vivo and in vitro. We classified and screened the LD4-PDT proteomic data to obtain key targets. Proteomic data revealed that EPHX2 and STAT3 are key targets of LD4-PDT for UC. Moreover, transcription factor STAT3 positively regulates the expression of EPHX2. Inhibiting EPHX2 can prevent the activation of NF-κB signaling pathway. Next, through pharmacological inhibition experiments, we confirmed that LD4-PDT can reduce intestinal inflammation by inhibiting STAT3-EPHX2 axis. However, by treating normal intestinal epithelial cells and colon cancer cells with TPPU and Stattic, our data confirmed that the STAT3-EPHX2 axis does not exist in colon cancer. In this study, we demonstrated that the transcription factor STAT3 can positively regulate the expression of EPHX2 in normal colon. LD4 can alleviate UC by inhibiting the STAT3-EPHX2 axis, but this axis does not exist in colon cancer. LD4-PDT may become a new and effective method for treating UC.

Design, synthesis and biological evaluation of novel naphthoquinothiazole derivatives as potent antitumor agents through inhibiting STAT3.

The signal transducer and activator of transcription 3 (STAT3) has been established as a crucial drug target in the development of antitumor agents. In this study, a series of 21 derivatives of the STAT3 inhibitor napabucasin were designed and synthesized. Through preliminary screening against tumor cell lines, SZ6 emerged as the most potent compound with half maximal inhibitory concentration (IC50) values of 46.3 nM, 66.4 nM, and 53.8 nM against HCT116, HepG2, and Hela cells respectively. Furthermore, SZ6 effectively suppressed tumor invasion and migration in HCT116 cell assays by inducing S-phase arrest and apoptosis through inhibition of Protein Kinase B (PKB/AKT) activity and induction of reactive oxygen species (ROS). The mechanism underlying SZ6's action involves inhibition of STAT3 phosphorylation, which was confirmed by western blotting analysis. Additionally, surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA) demonstrated direct binding between SZ6 and STAT3. Notably, in vivo studies revealed that SZ6 significantly inhibited tumor growth without any observed organ toxicity. Collectively, these findings identify SZ6 as a promising STAT3 inhibitor for colorectal cancer treatment.

Baicalein triggers ferroptosis in colorectal cancer cells via blocking the JAK2/STAT3/GPX4 axis.

Colorectal cancer (CRC) is a prevalent form of gastrointestinal malignancy with challenges in chemotherapy resistance and side effects. Effective and low toxic drugs for CRC treatment are urgently needed. Ferroptosis is a novel mode of cell death, which has garnered attention for its therapeutic potential against cancer. Baicalein (5, 6, 7-trihydroxyflavone) is the primary flavone extracted from the dried roots of Scutellaria baicalensis that exhibits anticancer effects against several malignancies including CRC. In this study, we investigated whether baicalein induced ferroptosis in CRC cells. We showed that baicalein (1-64 µM) dose-dependently inhibited the viability of human CRC lines HCT116 and DLD1. Co-treatment with the ferroptosis inhibitor liproxstatin-1 (1 µM) significantly mitigated baicalein-induced CRC cell death, whereas autophagy inhibitor chloroquine (25 µM), necroptosis inhibitor necrostatin-1 (10 µM), or pan-caspase inhibitor Z-VAD-FMK (10 µM) did not rescue baicalein-induced CRC cell death. RNA-seq analysis confirmed that the inhibitory effect of baicalein on CRC cells is associated with ferroptosis induction. We revealed that baicalein (7.5-30 µM) dose-dependently decreased the expression levels of GPX4, key regulator of ferroptosis, in HCT116 and DLD1 cells by blocking janus kinase 2 (JAK2)/STAT3 signaling pathway via direct interaction with JAK2, ultimately leading to ferroptosis in CRC cells. In a CRC xenograft mouse model, administration of baicalein (10, 20 mg/kg, i.g., every two days for two weeks) dose-dependently inhibited the tumor growth with significant ferroptosis induced by inhibiting the JAK2/STAT3/GPX4 axis in tumor tissue. This study demonstrates that ferroptosis contributes to baicalein-induced anti-CRC activity through blockade of the JAK2/STAT3/GPX4 signaling pathway, which provides evidence for the therapeutic application of baicalein against CRC.

Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

Evaluation of STAT3 Inhibition by Cancer Chemopreventive Trichothecenes Derived from Metabolites of Trichothecium roseum.

This study evaluated the ability of isolated or semisynthesized trichothecene sesquiterpenes to prevent cancer emergence and proliferation and inhibit signal transducer and activator of transcription-3 (STAT3) phosphorylation through in vitro assays. Trichothecinol A (TTC-A), which bears a hydroxy group at C3, exhibited greater cancer prevention, antiproliferation, and STAT3 phosphorylation inhibition effects than trichothecin (TTC), which lacks a hydroxy group at C3. Furthermore, trichothecinol B (TTC-B), which is a reduced derivative of TTC and has similar cytotoxic effect, showed substantially weaker chemoprotection and STAT3 phosphorylation inhibition effects than TTC. These results clearly indicate that the hydroxy group at C3 and carbonyl group at C8 are crucial for inducing both potent chemoprevention and STAT3 phosphorylation inhibition.

Synthesis, Antiproliferative Activity and Molecular Docking Analysis of Both Enantiomerically Pure Decursin Derivatives as Anticancer Agents.

Using (S)-decursinol isolated from root of Angelica gigas Nakai (AGN), we semi-synthesized and evaluated a series of both enantiomerically pure decursin derivatives for their antiproliferative activities against A549 human lung cancer cells. All synthesized compounds showed a broad spectrum of inhibitory activities against the growth of A549 cells. Especially, compound (S)-2d with (E)-(furan-3-yl)acryloyl group showed the most potent activity (IC50: 14.03 µM) against A549 cancer cells as compared with the reference compound, decursin (IC50: 43.55 µM) and its enantiomer, (R)-2d (IC50: 151.59 µM). Western blotting assays indicated that (S)-2d more strongly inhibited Janus kinase 1 (JAK1) and signal transducer and activator of transcription activation 3 (STAT3) phosphorylation than decursin in a dose-dependent manner, while having no effect on CXCR7 overexpression and total STAT3 level. In addition, (S)-2d induced cell cycle arrest at G1 phase and subsequent apoptotic cell death in A549 cancer cells. Our combined analysis of molecular docking studies and biological data suggests that the inhibition of JAK1 with (S)-2d resulted in loss of STAT3 phosphorylation and inhibition of cell growth in A549 cancer cells. These overall results strongly suggest that (S)-2d (MRC-D-004) as a novel JAK1 inhibitor may have therapeutic potential in the treatment of A549 human lung cancers by targeting the JAK1/STAT3 signaling pathway.

Microalgae Octapeptide IIAVEAGC Alleviates Oxidative Stress and Neurotoxicity in 6-OHDA-Induced SH-SY5Y Cells by Regulating the Nrf2/HO-1and Jak2/Stat3 Pathways.

Neurodegenerative diseases are characterized by the progressive loss of selectively vulnerable populations of neurons, and many factors are involved in its causes. Neurotoxicity and oxidative stress, are the main related factors. The octapeptide Ile-Ile-Ala-Val-Glu-Ala-Gly-Cys (IEC) was identified from the microalgae Isochrysis zhanjiangensis and exhibited potential anti-oxidative stress activity. In this study, the stability of α-synaptic protein binding to IEC was modeled using molecular dynamics, and the results indicated binding stabilization within 60 ns. Oxidative stress in neurons is the major cause of α-synaptic protein congestion. Therefore, we next evaluated the protective effects of IEC against oxidative stress and neurotoxicity in 6-ohdainduced Parkinson's disease (PD) model SH-SY5Y cells in vitro. In oxidative stress, IEC appeared to increase the expression of the antioxidant enzymes HO-1 and GPX through the antioxidant pathway of Nrf2, and molecular docking of IEC with Nrf2 and GPX could generate hydrogen bonds. Regarding apoptosis, IEC protected cells by increasing the Bcl-2/Bax ratio, inhibiting the caspase cascade, acting on p53, and modulating the Jak2/Stat3 pathway. The results indicated that IEC exerted neuroprotective effects through the inhibition of α-synaptic protein aggregation and antioxidant activity. Therefore, microalgal peptides have promising applications in the prevention and treatment of neurodegenerative diseases.

STAT3 phosphorylation inhibitor Bt354 exhibits anti-neoplastic activity in glioblastoma multiforme cells.

The high mortality rate of glioblastoma multiforme (GBM), a lethal primary brain tumor, is attributable to postsurgical recurrence. STAT3, an oncogenic protein, is a signal transducer and transcription activator encourages cancer cell migration and proliferation, which results in resistance to therapy. STAT3 inhibition reduces cancer metastasis and improves patient prognosis. Bt354, a small molecule STAT inhibitor, exhibits significant cytotoxic and anti-proliferative activities against certain cancer types. Here, we demonstrated that exposure of GBM cells (U87 MG) to Bt354 had a significant, concentration-dependent growth suppression. Bt354 also induced apoptosis and downregulated the expression of the epithelial-mesenchymal transition genes. Therefore, this study suggests the potential of Bt354 for treating GBM owing to its ability to induce cytotoxicity.

STAT3 inhibition ameliorates renal interstitial inflammation in MRL/lpr mice with diffuse proliferative lupus nephritis.

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) is one of the most common and severe clinical syndromes of diffuse proliferative lupus nephritis (DPLN), of which poor prognosis is indicated by aggravated renal function deterioration. However, the specific therapy and mechanisms of AKI in DPLN remain to be explored. METHODS: The correlation between AKI and clinical pathological changes in DPLN patients was analyzed. Expression of STAT3 signaling was detected in MRL/lpr mice with DPLN using immunohistochemical staining and immunoblotting. Inhibition of STAT3 activation by combination therapy was assessed in MRL/lpr mice. RESULTS: Correlation analysis revealed only the interstitial leukocytes were significantly related to AKI in endocapillary DPLN patients. MRL/lpr mice treated with vehicle, which can recapitulate renal damages of DPLN patients, showed upregulation of STAT3, pSTAT3 and caspase-1 in renal cortex. FLLL32 combined with methylprednisolone therapy significantly inhibited the STAT3 activation, improved acute kidney damage, reduced the interstitial infiltration of inflammatory cells and decreased the AKI incidence in MRL/lpr mice. CONCLUSION: STAT3 activation may play an important role in the pathogenesis of DPLN and the development of AKI. Hence, STAT3 inhibition based on the combination of FLLL32 with methylprednisolone may represent a new strategy for treatment of DPLN with AKI.

A novel p55PIK signaling peptide inhibitor alleviates neuroinflammation via the STAT3/NF-kB signaling pathway in experimental stroke.

BACKGROUND: Ischemic stroke remains the predominant contributor to mortality and disability globally. Microglia undergo rapid activation and initiate inflammatory cascade reactions by phenotypic polarization, participating in the regulation of inflammatory injury and tissue repair post-ischemic stroke. Regulating microglia-mediated neuroinflammation is a promising therapeutic strategy for ischemic stroke. Previously, we designed and synthesized a novel p55PIK inhibitor, TAT-N15 polypeptide, which presents inhibitive activity on NF-κB signaling-mediated inflammation in acute conjunctivitis and allergic rhinitis. The present study aimed to explore the therapeutic effect and mechanism of TAT-N15 on ischemia stroke. METHODS: The mouse model of transient cerebral ischemia was made using the intraluminal filament method. After being treated with daily intraperitoneal injections of TAT-N15 (10 mg/kg) for 7 d, the neurological outcomes and the cerebral infarction volume were evaluated. Histopathology of the ischemia cerebral hemisphere was observed by H&E and Nissl staining. Neuronal survival, astrogliosis, and co-labeling of CD86/Iba1 and CD206/Iba1 were detected by immunofluorescence. The cell apoptosis was estimated by TUNEL staining. The expression levels of apoptosis-associated proteins, proinflammatory cytokines, protein markers of M1 and M2 microglia, and the phosphorylation of NF-κB and STAT3 proteins in the ischemic penumbra were detected by Western blot. RESULTS: TAT-N15 treatment significantly decreased the infarct volume and alleviated neurological functional impairment, neuronal injury, and neuron apoptosis. Meanwhile, TAT-N15 treatment restrained the activation of microglia and astrocytes as well as the protein expression of proinflammatory cytokine in ischemic penumbra. Additionally, the administration of TAT-N15 treatment resulted in a significant reduction in the density of M1 phenotype microglia while concurrently increasing the density of M2 phenotype microglia within the ischemic penumbra. Finally, mechanical analysis unveiled that TAT-N15 exerted a substantial inhibitory effect on the protein expression of phosphorylated STAT3 and NF-κB. CONCLUSION: TAT-N15 may inhibit neuroinflammation via regulating microglia activation and polarization through the STAT3/NF-κB pathway, which exhibits the neuroprotection effect in ischemic stroke.

The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase.

Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Endoplasmic reticulum stress promotes sepsis-induced muscle atrophy via activation of STAT3 and Smad3.

Endoplasmic reticulum (ER) stress is involved in skeletal muscle atrophy in various conditions, but the role of ER stress in sepsis-induced muscle atrophy is not well understood. In this study, we conducted experiments in wild-type (WT) mice and C/EBP homologous protein knockout (CHOP KO) mice to explore the role and mechanism of ER stress in sepsis-induced muscle atrophy. Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis. In WT mice, the body weight, muscle mass, and cross-sectional area of muscle fibers in CLP group both decreased significantly compared with sham group, which revealed that sepsis-induced dramatic muscle atrophy. Additionally, sepsis activated the ubiquitin-proteasome system (UPS), accompanied by the activation of ER stress. In vitro, inhibition of ER stress suppressed the activity of E3 ubiquitin ligases and alleviated the myotube atrophy. In vivo, CHOP KO also reduced the expression of E3 ubiquitin ligases and UPS-mediated protein degradation, and significantly attenuated sepsis-induced muscle atrophy. Deletion of CHOP also decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and Smad3, and inhibition of STAT3 and Smad3 partly reduced proteolysis caused by ER stress in vitro. These findings confirm that ER stress activates UPS-mediated proteolysis and promotes sepsis-induced muscle atrophy, which is partly achieved by activating STAT3 and Smad3.

Mosaic IL6ST variant inducing constitutive GP130 cytokine receptor signaling as a cause of neonatal onset immunodeficiency with autoinflammation and dysmorphy.

Interleukin-6 signal transducer (IL6ST) encodes the GP130 protein which transduces the proinflammatory signaling of the IL6 cytokine family through Janus kinase signal transducers and activators of transcription pathway (JAK/STAT) activation. Biallelic loss-of-function IL6ST variants cause autosomal recessive hyper-IgE syndrome or a variant of the Stuve-Wiedemann syndrome. Somatic gain-of-function IL6ST mutations, in particular, small monoallelic in-frame deletions of which the most prevalent is the IL6ST Ser187_Tyr190del, are an established cause of inflammatory hepatocellular tumors, but so far, no disease caused by such mutations present constitutively has been described. Herein, we report a pediatric proband with a novel syndrome of neonatal onset immunodeficiency with autoinflammation and dysmorphy associated with the IL6ST Tyr186_Tyr190del variant present constitutively. Tyr186_Tyr190del was found by exome sequencing and was shown to be de novo (absent in proband's parents and siblings) and mosaic (present in approximately 15-40% of cells depending on the tissue studied-blood, urine sediment, hair bulbs and buccal swab). Functional studies were performed in the Epstein-Barr virus-immortalized patient's B cell lymphoblastoid cell line, which carried the variant in approximately 95% of the cells. Western blot showed that the patient's cells exhibited constitutive hyperphosphorylation of Tyr705 in STAT3, which is indicative of IL6-independent activation of GP130. Interestingly, the STAT3 phosphorylation could be inhibited with ruxolitinib as well as tofacitinib, which are clinically approved JAK1 and JAK3 (to lesser extent JAK2 and JAK1) inhibitors, respectively. Given our results and the recent reports of ruxolitinib and tofacitinib use for the treatment of diseases caused by direct activation of STAT3 or STAT1, we speculate that these drugs may be effective in the treatment of our patient's condition.

The stem-like Stat3-responsive cells of zebrafish intestine are Wnt/ß-catenin dependent.

The transcription factor Stat3 is required for proliferation and pluripotency of embryonic stem cells; we have prepared and characterized fluorescent Stat3-reporter zebrafish based on repeats of minimal responsive elements. These transgenic lines mimic in vivo Stat3 expression patterns and are responsive to exogenous Stat3; notably, fluorescence is inhibited by both stat3 knockout and IL6/Jak/STAT inhibitors. At larval stages, Stat3 reporter activity correlates with proliferating regions of the brain, haematopoietic tissue and intestine. In the adult gut, the reporter is active in sparse proliferating cells, located at the base of intestinal folds, expressing the stemness marker sox9b and having the morphology of mammalian crypt base columnar cells; noteworthy, zebrafish stat3 mutants show defects in intestinal folding. Stat3 reporter activity in the gut is abolished with mutation of T cell factor 4 (Tcf7l2), the intestinal mediator of Wnt/ß-catenin-dependent transcription. The Wnt/ß-catenin dependence of Stat3 activity in the gut is confirmed by abrupt expansion of Stat3-positive cells in intestinal adenomas of apc heterozygotes. Our findings indicate that Jak/Stat3 signalling is needed for intestinal stem cell maintenance and possibly crucial in controlling Wnt/ß-catenin-dependent colorectal cancer cell proliferation.

Antagonism of STAT3 signalling by Ebola virus.

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer.