STAT3-dependent Transcription Research Articles

#2804 Mechanisms of cAMP-induced cyst formation observed in Pkhd1-KO cells

Abstract Background and Aims Loss of ciliary protein function leads to altered epithelial properties in hereditary polycystic kidney diseases. To address molecular aspects of defective epithelial homeostasis, monolayered epithelial spheroids can be used to analyze lack of protein function and consequences of pharmacological intervention. Here, we employed epithelial cell clones deficient for fibrocystin/polyductin (FPC) protein function, the origin of ARPKD, to study the impact of FPC cytoplasmic domain constructs on cAMP-induced cystogenesis. Method We used pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout (KO) of Pkhd1 / FPC, and corresponding controls. Cells were grown in matrigel to allow formation of epithelial spheroids within 3 days. Forskolin (Fsk) treatment was employed to induce cAMP-mediated cyst growth mimicking disease conditions. Measurement of proportional lumen, the ratio of lumen to spheroid area, allowed detection the enhanced water and ion transport across the barrier as characteristic for secretory epithelia. Cystogenic signals were modulated by expression of the FPC C-terminal domain constructs and/or pharmacological inhibitors, and involvement of ion channels was addressed. Results In Pkhd1-KO cell lines, enhanced cAMP levels resulted in massive lumen expansion of epithelial spheroids with no increase in cell number. The rise in proportional lumen was sensitive to inhibition of chloride-channels, CFTR and TMEM16A, and Src kinase. Fsk-mediated cyst induction led to activation and increased Y705 phosphorylation of STAT3. To address the impact of FPC on cystogenic signaling, expression of a membrane-bound FPC C-terminal protein domain was studied, and its processing and intracellular localization determined. Controlled expression of the FPC cytoplasmic domain in Pkhd1-KO cell lines suppressed Fsk-induced cyst formation. In addition, the FPC domain reduced activation of Src and STAT3, leading to lower STAT3-dependent transcription. Conclusion In FPC-deficient pl-MDCK cells, cyst formation stimulated by high cAMP levels is associated with enhanced Src/STAT3 signaling. Expression of the FPC cytoplasmic domain, leading to a gain-of-function, suppresses Src and STAT3 activity in Pkhd1-KO epithelia and limits cyst growth in vitro. Control of cystogenic Src/STAT3 signaling can be considered a physiologic function of the FPC protein.

Dexamethasone and OLT1177 Cooperate in the Reduction of Melanoma Growth by Inhibiting STAT3 Functions.

The NLRP3 inflammasome is a multimolecular complex that processes inactive IL-1β and IL-18 into proinflammatory cytokines. OLT1177 is an orally active small compound that specifically inhibits NLRP3. Here, B16F10 melanoma were implanted in mice and treated with OLT1177 as well as combined with the glucocorticoid dexamethasone. At sacrifice, OLT1177 treated mice had significantly smaller tumors compared to tumor-bearing mice treated with vehicle. However, the combined treatment of OLT1177 plus dexamethasone revealed a greater suppression of tumor growth. This reduction was accompanied by a downregulation of nuclear and mitochondrial STAT3-dependent gene transcription and by a significant reduction of STAT3 Y705 and S727 phosphorylations in the tumors. In vitro, the human melanoma cell line 1205Lu, stimulated with IL-1α, exhibited significantly lower levels of STAT3 Y705 phosphorylation by the combination treatment, thus affecting the nuclear functions of STAT3. In the same cells, STAT3 serine 727 phosphorylation was also lower, affecting the mitochondrial functions of STAT3. In addition, metabolic analyses revealed a marked reduction of ATP production rate and glycolytic reserve in cells treated with the combination of OLT1177 plus dexamethasone. These findings demonstrate that the combination of OLT1177 and dexamethasone reduces tumor growth by targeting nuclear as well as mitochondrial functions of STAT3.

Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation.

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.

NOTCH4 Exhibits Anti-Inflammatory Activity in Activated Macrophages by Interfering With Interferon-γ and TLR4 Signaling.

NOTCH4 is a member of the NOTCH family of receptors whose expression is intensively induced in macrophages after their activation by Toll-like receptors (TLR) and/or interferon-γ (IFN-γ). In this work, we show that this receptor acts as a negative regulator of macrophage activation by diminishing the expression of proinflammatory cytokines, such as IL-6 and IL-12, and costimulatory proteins, such as CD80 and CD86. We have observed that NOTCH4 inhibits IFN-γ signaling by interfering with STAT1-dependent transcription. Our results show that NOTCH4 reprograms the macrophage response to IFN-γ by favoring STAT3 versus STAT1 phosphorylation without affecting their expression levels. This lower activation of STAT1 results in diminished transcriptional activity and expression of STAT1-dependent genes, including IRF1, SOCS1 and CXCL10. In macrophages, NOTCH4 inhibits the canonical NOTCH signaling pathway induced by LPS; however, it can reverse the inhibition exerted by IFN-γ on NOTCH signaling, favoring the expression of NOTCH-target genes, such as Hes1. Indeed, HES1 seems to mediate, at least in part, the enhancement of STAT3 activation by NOTCH4. NOTCH4 also affects TLR signaling by interfering with NF-κB transcriptional activity. This effect could be mediated by the diminished activation of STAT1. These results provide new insights into the mechanisms by which NOTCH, TLR and IFN-γ signal pathways are integrated to modulate macrophage-specific effector functions and reveal NOTCH4 acting as a new regulatory element in the control of macrophage activation that could be used as a target for the treatment of pathologies caused by an excess of inflammation.

Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability.

ABSTRACTVascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, including cancer, stroke, cardiovascular disease, retinal conditions and COVID-19-associated pulmonary edema, sepsis and acute lung injury. Understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics to inhibit vascular permeability, while preserving tissue-restorative angiogenesis. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. We show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9-generated Stat3 knockout zebrafish. Intercellular adhesion molecule 1 (ICAM-1) expression is transcriptionally regulated by STAT3, and VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse and human endothelium. Collectively, our findings suggest that VEGF/VEGFR-2/JAK2/STAT3 signaling regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability. This article has an associated First Person interview with the first author of the paper.

Y705 and S727 are required for the mitochondrial import and transcriptional activities of STAT3, and for regulation of stem cell proliferation.

ABSTRACTThe STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that import of STAT3 inside mitochondria requires Y705 phosphorylation by Jak, whereas its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: although the Y705-mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect import into the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription appears to be independent from STAT3 binding to STAT3-responsive elements. Finally, loss-of-function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.

Th17 Cells Targeting By MiR-21 Inhibitor Impairs Multiple Myeloma Related Bone Disease

Bone disease (BD) represents a hallmark of multiple myeloma (MM), characterized by classical osteolytic lesions and/or osteoporosis, that can be life threatening and contribute to poor outcomes. The complex interplay among MM conditioned bone effectors such as MM cells, osteoclasts (OCLs) and osteoblasts within BM microenvironment (BMM) supports MMBD. Several studies have shown that the extent of BD is tightly correlated to Th17 cells development and maintenance. Th17 secrete pro OCL cytokines such as RANKL, TNF, and IL-17, contributing to MMBD development. Recent findings showed that microRNAs (miRNAs) are involved in MMBD. MiRNAs are short non-coding RNAs that regulate gene expression at a post transcriptional level. Among others, miR-21 is involved in Th17 differentiation by targeting SMAD-7, a negative regulator of TGF-β signaling, promoting the activation of SMAD-2/3, suppressing IL-2 expression, and ultimately enhancing the activity of the TGF-β signaling pathway. Taken together, these data support a therapeutic strategy of miR-21 targeting to ameliorate MMBD through Th17 inhibition. We have previously demonstrated that miR-21 activity within BMM plays a crucial role in bone resorption/apposition balance through PIAS3 suppression and STAT3 dependent RANKL transcription in BM stromal cells. In the present study, we sought to evaluate the role of miR-21 in Th17 mediated BD. We enriched Th17 from BM CD4+CCR6+ T cells and evaluated miR-21 levels. We found increased miR-21 in MM derived Th17 (MMTh17) as compared to controls, supporting the relevance of miR-21 in MMTh17. To further explore the mechanisms of miR-21 activity in Th17 function, freshly isolated naïve CD4+ T cells were differentiated from healthy donors into Th17 along 18 days of culture in the presence of polarizing cytokines. At the end of culture, T cells expressed the Th17 full maturation markers RORC and IL17. As a model of MMBD in vitro, OCL resorptive activity was evaluated on dentine discs with/without Th17 freshly isolated from MM patients or differentiated in vitro. In both cases, Th17 promoted potent OCL mediated dentin pits generation as compared to controls. To counteract miR-21 activity, we electroporated naïve CD4+ T cells with miR-21 inhibitor (miR21i) or scramble control mirna before starting Th17 differentiation culture. MiR-21 expression levels were assessed by rt pcr before and at the end of differentiation culture to ascertain its effective inhibition. To map signaling pathways differences elicited by miR21i-Th17, we performed a mass spectrometry-based quantitative phosphoproteomics analysis. 1134 phosphorylated proteins were identified, with 386 significant changes as compared to controls. The global-pairwise-normalized data underwent to Ingenuity Pathway Analysis (IPA). Interestingly, analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition included PI3K/AKT and 14-3-3 Signaling. Among Th17 potential regulators, enhanced activity of PDCD4, STAT1, MINK1 and FOXO1 were observed, while ITGAL showed reduced activity. On these bases, we performed WB to validate IPA analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of RORC were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated in the presence of miR-21 antagonism. STAT3 is a master transcription factor for Th17 upon IL-6 stimulation. As PIAS3 and PDCA4 negatively regulate STAT3, we checked phospho-STAT3 expression and found it downregulated as expected. Overall, these data indicated that Th17 differentiation was impaired by miR-21i. In the second set of experiments, we aimed to evaluate Th17 functional impairment by miR-21 antagonism in MMBD. OCLs were plated with dentine discs and exposed to either Th17 alone, miR21i-Th17, or RANKL/MCSF cytokine alone as positive control. Dentine pits and areas were significantly lower in the presence of miR21i-Th17 as compared to Th17 alone and positive control (Fig.1). These data indicated that miR-21 antagonism negatively regulates Th17 mediated OCL activity and support the role of miR-21 inhibition as an innovative treatment tool in MMBD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PP2A-activating Drugs Enhance FLT3 Inhibitor Efficacy through AKT Inhibition-Dependent GSK-3β-Mediated c-Myc and Pim-1 Proteasomal Degradation.

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD-expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD-expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD-expressing cells through a novel mechanism involving AKT inhibition-dependent GSK-3β-mediated increased c-Myc and Pim-1 proteasomal degradation.

PIM Kinases Promote Survival and Immune Escape in Primary Mediastinal Large B-Cell Lymphoma through Modulation of JAK-STAT and NF-κB Activity

Primary mediastinal large B-cell lymphoma (PMBL) cells depend on the constitutive activity of NF-κB and STAT transcription factors, which drive expression of multiple molecules essential for their survival. In a molecularly related B-cell malignant tumor (classic Hodgkin lymphoma), tumor Reed-Sternberg cells overexpress oncogenic (proviral integration site for Moloney murine leukemia virus (PIM) 1, 2, and 3 kinases in a NF-κB- and STAT-dependent manner and PIMs enhance survival and expression of immunomodulatory molecules. Given the multiple overlapping characteristics of Reed-Sternberg and PMBL cells, we hypothesized that PIM kinases may be overexpressed in PMBL and involved in PMBL pathogenesis. The expression of PIM kinases in PMBL diagnostic biopsy specimens was assessed and their role in survival and immune escape of the tumor cells was determined. PIMs were abundantly expressed in primary tumors and PMBL cell lines. Inhibition of PIM kinases was toxic to PMBL cells, attenuated protein translation, and down-regulated NF-κB- and STAT-dependent transcription of prosurvival factors BCL2A1, BCL2L1, and FCER2. Furthermore, PIM inhibition decreased expression of molecules engaged in shaping the immunosuppressive microenvironment, including programmed death ligand 1/2 and chemokine (C-C motif) ligand 17. Taken together, our data indicate that PIMs support PMBL cell survival and immune escape and identify PIMs as promising therapeutic targets for PMBL.

Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death.

Because of its key role in cancer development and progression, STAT3 has become an attractive target for developing new cancer therapeutics. While several STAT3 inhibitors have progressed to advanced stages of development, their underlying biology and mechanisms of action are often more complex than would be expected from specific binding to STAT3. Here, we have identified and optimized a series of compounds that block STAT3-dependent luciferase expression with nanomolar potency. Unexpectedly, our lead compounds did not bind to cellular STAT3 but to another prominent anticancer drug target, TrxR1. We further identified that TrxR1 inhibition induced Prx2 and STAT3 oxidation, which subsequently blocked STAT3-dependent transcription. Moreover, previously identified inhibitors of STAT3 were also found to inhibit TrxR1, and likewise, established TrxR1 inhibitors block STAT3-dependent transcriptional activity. These results provide new insights into the complexities of STAT3 redox regulation while highlighting a novel mechanism to block aberrant STAT3 signaling in cancer cells.

A Clinical Trial of the STAT3 Inhibitor Pyrimethamine in Chronic Lymphocytic Leukemia

Despite advances in targeted therapy, chronic lymphocytic leukemia (CLL) remains a highly prevalent and essentially incurable malignancy. One molecular hallmark of CLL is the constitutive serine phosphorylation and transcriptional activation of the oncogenic transcription factor STAT3. Targeting STAT3 may have a high therapeutic index since normal cells can tolerate a loss of STAT3 function. To identify STAT3 inhibitors that could be rapidly introduced into proof-of-concept clinical trials, we screened a chemical library of drugs known to be safe in humans for specific inhibitors of STAT3-dependent transcription. Using this strategy, we identified the anti-parasitic agent pyrimethamine as a drug that could inhibit STAT3 at levels safely achieved for months at a time in humans. Pyrimethamine inhibited STAT3-dependent gene expression in CLL cells ex vivo, and decreased survival of CLL cells, but not peripheral blood mononuclear cells from healthy donors. To determine whether STAT3 inhibition would confer clinical benefit in patients with CLL, we conducted a phase I clinical trial of continuous daily pyrimethamine, in the era before targeted therapy, in relapsed CLL patients whose disease progressed despite standard therapies. We used a typical 3+3 dose escalation design with three cohorts, 12.5, 25 and 50 mg per day (mg/d). Samples for PK/PD analysis were drawn weekly in the first month and every other week in the second. Sixteen heavily pretreated patients enrolled on the phase 1 portion of this study. The median age was 63 (36-85) and the median time from diagnosis to study therapy was 74 months (range, 6-176 months). Six patients had 17p deletion and 4 had 11q deletion, while 12 of 13 evaluable patients had unmutated IGHV. The patients had a median of 6 prior therapies, including 8 patients who had received prior high dose methylprednisolone, five who had received prior alemtuzumab, and one each with prior allogeneic and autologous stem cell transplantation. Three patients each enrolled on cohorts 1 and 2, and ten patients enrolled on cohort 3. There were no dose limiting toxicities and no significant drug-related toxicities. No objective responses by IW-CLL 2008 criteria were observed. Half of patients achieved stable disease, with one patient dosed at 50 mg/d on therapy for 12 months, and two at 25 mg/d on therapy for 4 and 6 months. The remaining patients had progressive disease, and all but one patient discontinued therapy for progressive disease. The median time on therapy was 1.1 months (0.23-9.99), with median progression free survival of 1.5 months (0.92-5.52) and median overall survival of 22 months (11.75-NA).To determine whether the levels of pyrimethamine achieved in patients corresponded with levels known to inhibit STAT3 transcriptional function in vitro, we measured trough concentrations of pyrimethamine in plasma and in peripheral blood mononuclear cells at distinct time points for patients on the trial. Plasma concentrations and intracellular concentrations were highly correlated, and also correlated closely with dose level. Only in the highest dose cohort did the plasma concentrations reproducibly approach the threshold of 10 micromolar at which STAT3 inhibition occurred consistently in vitro.To determine whether pyrimethamine was exerting an on-target effect in inhibiting STAT3-dependent gene expression in vivo, we first defined a signature of five STAT3-dependent genes (AIM2, ATXN1, ENPP2, GAB1, and ID3) that are upregulated in CLL cells relative to normal B lymphocytes. We then determined expression of these genes from the patients' CLL cells at baseline and while on pyrimethamine. In addition, to further generate predictors of response, we also quantitated the cytotoxic effect of pyrimethamine on the pre-treatment cells of patients ex vivo. We then correlated these potential predictive and pharmacodynamic biomarkers with drug levels and clinical course.In conclusion, pyrimethamine is an inhibitor of STAT3 transcriptional function that can safely be given to patients with CLL. It may be necessary to increase the daily dose of pyrimethamine to beyond 50 mg/day to adequately determine whether this drug can exert an on-target therapeutic effect in CLL patients. Since STAT3 target genes include many immune-suppressive and anti-apoptotic genes, it is likely that pyrimethamine will be most beneficial when combined with other therapeutic modalities. DisclosuresBrown:Pharmacyclics: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Boehringer: Consultancy; Sun Pharmaceutical Industries: Research Funding; Gilead: Consultancy, Research Funding; Loxo: Consultancy; Verastem: Consultancy, Research Funding; Invectys: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy; Janssen: Consultancy; Acerta / Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; Genentech: Consultancy; Beigene: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy. Wu:Neon Therapeutics: Equity Ownership.

Toxoplasma gondii stabilises tetrameric complexes of tyrosine-phosphorylated signal transducer and activator of transcription-1 and leads to its sustained and promiscuous DNA binding.

Toxoplasma gondii is an obligate intracellular parasite that infects up to 30% of humans worldwide. It can lead to severe diseases particularly in individuals with immature or defective immune responses. Control of T.gondii relies on the IFN-γ-induced signal transducer and activator of transcription-1 (STAT1) pathway. T.gondii, however, largely inactivates STAT1-mediated gene transcription by T.gondii inhibitor of STAT1-dependent transcription (TgIST), a parasite effector protein binding to STAT1. Here, we have analysed requirements of STAT1 to bind TgIST and characterised downstream effects on STAT1 signalling. TgIST bound to STAT1 dimers but more efficiently assembled with STAT1 tetramers, which are essential for effective IFN-γ responsiveness. Such binding was abrogated in N-terminal, but not C-terminal deletion mutants of STAT1. Furthermore, TgIST did not bind to the STAT1F77A substitution mutant that cannot form STAT1 tetramers, resulting in a complete unresponsiveness of parasite-infected STAT1F77A -expressing cells to IFN-γ. Remarkably, binding of TgIST considerably increased the affinity of the aberrant STAT1 tetramers for DNA consensus sequence binding motifs and even enabled binding to nonconsensus sequences. Consistent with the increased DNA binding, STAT1 from parasite-infected cells remained phosphorylated at Tyr701 and Ser727 and was retained within the nucleus in a DNA-bound state. The sustained and promiscuous binding activity particularly of STAT1 tetramers to unspecific DNA sites lacking a consensus STAT1-binding motif is an as yet unrecognised mechanism contributing to the defective IFN-γ-mediated signalling in T.gondii-infected cells.

Pharmacologic inhibition of STAT5 in acute myeloid leukemia

The transcription factor STAT5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. STAT5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of acute myeloid leukemia (AML). Since STAT5 is a critical mediator of diverse malignant properties of AML cells, direct targeting of STAT5 is of significant clinical value. Here, we describe the development and preclinical evaluation of a novel, potent STAT5 SH2 domain inhibitor, AC-4–130, which can efficiently block pathological levels of STAT5 activity in AML. AC-4–130 directly binds to STAT5 and disrupts STAT5 activation, dimerization, nuclear translocation, and STAT5-dependent gene transcription. Notably, AC-4–130 substantially impaired the proliferation and clonogenic growth of human AML cell lines and primary FLT3-ITD+ AML patient cells in vitro and in vivo. Furthermore, AC-4–130 synergistically increased the cytotoxicity of the JAK1/2 inhibitor Ruxolitinib and the p300/pCAF inhibitor Garcinol. Overall, the synergistic effects of AC-4–130 with TK inhibitors (TKIs) as well as emerging treatment strategies provide new therapeutic opportunities for leukemia and potentially other cancers.

Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein.

Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects.

Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

A heterozygous mutation in the coiled-coil domain of STAT5B is associated with immune deficiency and dysfunction

Abstract The signal transducer and activator of transcription (STAT) family of transcription factors play a critical role in regulating gene expression required for immune development and function. In a patient diagnosed with primary immunodeficiency characterized by chronic lymphadenopathy, splenomegaly, immune thrombocyotopenia and necrotizing granulomas, we discovered a heterozygous missense mutation in STAT5B (c.617A>G, p.Q206R). Cotransfection of Q206R and wild-type STAT5B in HEK293 cells reduced STAT5B-mediated transcriptional activity compared to wild-type STAT5B alone, suggesting that Q206R STATB dominantly-interfered with wild-type STAT5B function. Patient T cell blasts exhibited reduced phosphorylation of STAT5B in response to IL-2, IL-7 and IL-15 compared to healthy controls. Further, IL-2/STAT5-dependent gene transcription was impaired in patient T cell blasts. The patient exhibited increased lymphocytes (as well as monocytes and neutrophils) in the peripheral blood associated with a higher frequency and number of CD4+ T effector memory cells (TEMs). Further, CD4+ TEMs isolated from the patient were resistant to restimulation-induced cell death compared to healthy control TEMs in vitro. Collectively, these data suggest that the Q206R STAT5B variant reduced IL-2/STAT5B-mediated gene expression in human T cells, and defects in this signaling pathway are associated with increased survival of TEMs, disrupted immune homeostasis and disease.

Activation of Male Liver Chromatin Accessibility and STAT5-Dependent Gene Transcription by Plasma Growth Hormone Pulses.

Sex differences in pituitary growth hormone (GH) secretion (pulsatile in males vs near continuous/persistent in females) impart sex-dependent expression to hundreds of genes in adult mouse liver. Signal transducer and activator of transcription (STAT) 5, a GH-activated transcription factor that is essential for liver sexual dimorphism, is dynamically activated in direct response to each male plasma GH pulse. However, the impact of GH-induced STAT5 pulses on liver chromatin accessibility and downstream transcriptional events is unknown. In this study, we investigated the impact of a single pulse of GH given to hypophysectomized mice on local liver chromatin accessibility (DNase hypersensitive site analysis), transcription rates (heterogeneous nuclear RNA analysis), and gene expression (quantitative polymerase chain reaction and RNA sequencing) determined 30, 90, or 240 minutes later. The STAT5-dependent but sex-independent early GH response genes Igf1 and Cish showed rapid, GH pulse-induced increases in chromatin accessibility and gene transcription, reversing the effects of hypophysectomy. Rapid increases in liver chromatin accessibility and transcriptional activity were also induced in hypophysectomized male mice for some (Ces2b, Ugt2b38) but not for other liver STAT5-dependent male-biased genes (Cyp7b1). Moreover, in pituitary-intact male mice, Igf1, Cish, Ces2b, and Ugt2b38 all showed remarkable cycles of chromatin opening and closing, as well as associated cycles of induced gene transcription, which closely followed each endogenous pulse of liver STAT5 activity. Thus, the endogenous rhythms of male plasma GH pulsation dynamically open and then close liver chromatin at discrete, localized regulatory sites in temporal association with transcriptional activation of Igf1, Cish, and a subset of STAT5-dependent male-biased genes.

The DEAD box protein p68: a novel coactivator of Stat3 in mediating oncogenesis.

DEAD box RNA helicase p68 acts as a transcriptional coactivator of several oncogenic transcription factors apart from being a vital player of RNA metabolism. Signal transducer and activator of transcription 3 (Stat3) is a major oncogenic contributor of diverse cancers, including that of colon. Deciphering the mechanistic insights of coactivation of Stat3 transcriptional activity may aid in improved therapeutic strategies. Here we report for the first time a novel mechanism of alliance between p68 and Stat3 in stimulating transcriptional activity of Stat3. Interestingly, we observed that the expression of p68 and Stat3 bears strong positive correlation and significant colocalization in normal and colon carcinoma patient samples. We demonstrated that p68 directly interacts with Stat3 in HEK293 cells as well as multiple colon cancer cell lines. Additionally, p68 positively modulated both mRNA and protein expression levels of Stat3 target genes; promoter activity of Stat3 target gene Mcl-1 in multiple colon cancer cell lines. Also, p68 occupied the promoters of multiple Stat3 target genes in enhancing Stat3-dependent transcription. Moreover, the strong positive correlation between the abundance of p68 and Stat3 target genes in the same set of colon carcinoma samples further supported our observations. Enhanced expression levels of Stat3 target genes observed in primary tumors and metastatic lung nodules, generated in mice colorectal allograft model using syngeneic cells stably expressing p68, further reinforced our in vitro findings. Hence, this study unravels novel modes of p68-mediated oncogenesis through coactivation of Stat3 and enhancing Stat3 signaling.

The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities, motifs and genomic positions.

STAT5 interacts with other factors to control transcription, and the mechanism of regulation is of interest as constitutive active STAT5 has been reported in malignancies. Here, LSD1 and HDAC3 were identified as novel STAT5a interacting partners in pro-B cells. Characterization of STAT5a, LSD1 and HDAC3 target genes by ChIP-seq and RNA-seq revealed gene subsets regulated by independent or combined action of the factors and LSD1/HDAC3 to play dual role in their activation or repression. Genes bound by STAT5a alone or in combination with weakly associated LSD1 or HDAC3 were enriched for the canonical STAT5a GAS motif, and such binding induced activation or repression. Strong STAT5 binding was seen more frequently in intergenic regions, which might function as distal enhancer elements. Groups of genes bound weaker by STAT5a and stronger by LSD1/HDAC3 showed an absence of the GAS motif, and were differentially regulated based on their genomic binding localization and binding affinities. These genes exhibited increased binding frequency in promoters, and in conjunction with the absence of GAS sites, the data indicate a requirement for stabilization by additional factors, which might recruit LSD1/HDAC3. Our study describes an interaction network of STAT5a/LSD1/HDAC3 and a dual function of LSD1/HDAC3 on STAT5-dependent transcription, defined by protein–protein interactions, genomic binding localization/affinity and motifs.

A20 inhibition of STAT1 expression in myeloid cells: a novel endogenous regulatory mechanism preventing development of enthesitis

ObjectivesA20 is an important endogenous regulator of inflammation. Single nucleotide polymorphisms in A20 have been associated with various immune-mediated inflammatory diseases, and cell-specific deletion of A20 results in diverse inflammatory...