Role Of STAT3 Research Articles

STAT5 Is Necessary for the Metabolic Switch Induced by IL-2 in Cervical Cancer Cell Line SiHa.

The tumor cells reprogram their metabolism to cover their high bioenergetic demands for maintaining uncontrolled growth. This response can be mediated by cytokines such as IL-2, which binds to its receptor and activates the JAK/STAT pathway. Some reports show a correlation between the JAK/STAT pathway and cellular metabolism, since the constitutive activation of STAT proteins promotes glycolysis through the transcriptional activation of genes related to energetic metabolism. However, the role of STAT proteins in the metabolic switch induced by cytokines in cervical cancer remains poorly understood. In this study, we analyzed the effect of IL-2 on the metabolic switch and the role of STAT5 in this response. Our results show that IL-2 induces cervical cancer cell proliferation and the tyrosine phosphorylation of STAT5. Also, it induces an increase in lactate secretion and the ratio of NAD+/NADH, which suggest a metabolic reprogramming of their metabolism. When STAT5 was silenced, the lactate secretion and the NAD+/NADH ratio decreased. Also, the expression of HIF1α and GLUT1 decreased. These results indicate that STAT5 regulates IL-2-induced cell proliferation and the metabolic shift to aerobic glycolysis by regulating genes related to energy metabolism. Our results suggest that STAT proteins modulate the metabolic switch in cervical cancer cells to attend to their high demand of energy required for cell growth and proliferation.

Abstract 256: A novel transcriptional complex of OLIG2-STAT5 mediates glioma stem cell self renewal and invasion

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion. Citation Format: Aunay Miller, Costanza Lo Cascio, Mylan Blomquist, James B. McNamara, Keely Orndorff, Matthew Dufault, Christopher Sereduk, Joseph Loftus, Shwetal Mehta, Nhan L. Tran. A novel transcriptional complex of OLIG2-STAT5 mediates glioma stem cell self renewal and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 256.

STEM-15. STAT5-OLIG2 TRANSCRIPTIONAL COMPLEX MEDIATES GLIOMA STEM CELL SELF RENEWAL AND INVASION

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion.

Transcription factor STAT enhanced antimicrobial activities in Bombyx mori

STAT, a transcription factor in the JAK/STAT signaling pathway, regulates immune response to pathogens. In the silkworm (Bombyx mori), STAT exists as two split-forms, STAT-S and STAT-L. However, the role of STAT in silkworm immunity remains unclear. Our purpose was to investigate the effect of STAT on the expression of antimicrobial peptide (AMP) genes and resistance against pathogens. The expression levels of STAT-S and STAT-L were significantly up-regulated after induction by pathogenic microorganisms. In BmE cells, lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan stimulated STAT-S and STAT-L to transfer from the cytoplasm to the nucleus. We found that overexpression of STAT-S and STAT-L in cells could promote the expression of AMPs. We generated transgenic silkworm lines overexpressing STAT-L or STAT-S (OE-STAT-S; OE-STAT-L) or interfering with STAT (A4-dsSTAT). Overexpression of STAT-S and STAT-L upregulated the expression of AMP genes in the OE-STAT-S and OE-STAT-L, increased the survival rates of the OE-STAT-S silkworms and lowered the mortality of OE-STAT-L silkworms infected with S. aureus or Beauveria bassiana. By contrast, the death rate of A4-dsSTAT silkworms was higher after infection with these pathogenic microorganisms. These findings may provide insights into the role of STAT in the antimicrobial immune response of silkworms.

STAT1 is essential for HSC function and maintains MHCIIhi stem cells that resist myeloablation and neoplastic expansion

AbstractAdult hematopoietic stem cells (HSCs) are predominantly quiescent and can be activated in response to acute stress such as infection or cytotoxic insults. STAT1 is a pivotal downstream mediator of interferon (IFN) signaling and is required for IFN-induced HSC proliferation, but little is known about the role of STAT1 in regulating homeostatic hematopoietic stem/progenitor cells (HSPCs). Here, we show that loss of STAT1 altered the steady state HSPC landscape, impaired HSC function in transplantation assays, delayed blood cell regeneration following myeloablation, and disrupted molecular programs that protect HSCs, including control of quiescence. Our results also reveal STAT1-dependent functional HSC heterogeneity. A previously unrecognized subset of homeostatic HSCs with elevated major histocompatibility complex class II (MHCII) expression (MHCIIhi) displayed molecular features of reduced cycling and apoptosis and was refractory to 5-fluorouracil–induced myeloablation. Conversely, MHCIIlo HSCs displayed increased megakaryocytic potential and were preferentially expanded in CALR mutant mice with thrombocytosis. Similar to mice, high MHCII expression is a feature of human HSCs residing in a deeper quiescent state. Our results therefore position STAT1 at the interface of stem cell heterogeneity and the interplay between stem cells and the adaptive immune system, areas of broad interest in the wider stem cell field.

STAT1 mediates the PI3K/AKT pathway through promoting microRNA-18a in nasal polyps

Background Epithelial to mesenchymal transition (EMT) is linked to the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP). The involvement of STAT1 has been reported in CRSwNP. However, its specific role in regulating EMT in CRSwNP is not clear. We sought to evaluate the role of STAT1 in EMT in CRSwNP using clinical samples and a murine model. Methods Comprehensive analysis of differentially expressed genes was performed in nasal polyps from the CRSwNP patients, followed by pathway enrichment analysis. After bioinformatics prediction, the relationships between microRNA-18a (miR-18a) and PTEN or STAT1 were examined using dual-luciferase and RIP assays, respectively. The expression of STAT1, PTEN, and miR-18a in nasal tissues was detected using RT-qPCR, immunohistochemistry, and in situ hybridization. After the alteration of gene expression in mice with CRSwNP, western blot, RT-qPCR, and HE staining were conducted to detect EMT-related proteins, inflammatory factor secretion, inflammatory cell infiltration, and the PI3K/AKT pathway activity in nasal tissues. Results STAT1 and miR-18a were highly expressed, and PTEN was poorly expressed in the nasal polyp. STAT1 promoted transcription of miR-18a, which targeted PTEN. Downregulation of STAT1 and miR-18a inhibited the EMT and inflammatory cell infiltration, while depletion of PTEN promoted the EMT and inflammatory cell infiltration in the nasal polyp. The PI3K/AKT pathway was activated in the nasal polyp and regulated by the STAT1/miR-18a/PTEN axis. Conclusions STAT1 acts as a transcription factor to promote transcription of miR-18a, and miR-18a targets PTEN to exacerbate the inflammatory response and EMT in CRSwNP.

STAT6/VDR Axis Mitigates Lung Inflammatory Injury by Promoting Nrf2 Signaling Pathway.

Lung inflammatory injury is a global public health concern. It is characterized by infiltration of diverse inflammatory cells and thickening of pulmonary septum along with oxidative stress to airway epithelial cells. STAT6 is a nuclear transcription factor that plays a crucial role in orchestrating the immune response, but its function in tissue inflammatory injury has not been comprehensively studied. Here, we demonstrated that STAT6 activation can protect against particle-induced lung inflammatory injury by resisting oxidative stress. Specifically, genetic ablation of STAT6 was observed to worsen particle-induced lung injury mainly by disrupting the lungs' antioxidant capacity, as reflected by the downregulation of the Nrf2 signaling pathway, an increase in malondialdehyde levels, and a decrease in glutathione levels. Vitamin D receptor (VDR) has been previously proved to positively regulate Nrf2 signals. In this study, silencing VDR expression in human bronchial epithelial BEAS-2B cells consistently suppressed autophagy-mediated activation of the Nrf2 signaling pathway, thereby aggravating particle-induced cell damage. Mechanically, STAT6 activation promoted the nuclear translocation of VDR, which increased the transcription of autophagy-related genes and induced Nrf2 signals, and silencing VDR abolished these effects. Our research provides important insights into the role of STAT6 in oxidative damage and reveals its potential underlying mechanism. This information not only deepens the appreciation of STAT6 but also opens new avenues for the discovery of therapies for inflammatory respiratory system disorders.

STAT5 ablation in AgRP neurons increases female adiposity and blunts food restriction adaptations

AgRP neurons are important players in the control of energy homeostasis and are responsive to several hormones. In addition, STAT5 signalling in the brain, which is activated by metabolic hormones and growth factors, modulates food intake, body fat and glucose homeostasis. Given that, and the absence of studies that describe STAT5 function in AgRP cells, the present study investigated the metabolic effects of Stat5a/b gene ablation in these neurons. We observed that STAT5 signalling in AgRP neurons regulates body fat in female mice. However, male and female STAT5-knockout mice did not exhibit altered food intake, energy expenditure or glucose homeostasis compared to control mice. The counter-regulatory response or glucoprivic hyperphagia induced by 2-deoxy-d-glucose treatment were also not affected by AgRP-specific STAT5 ablation. However, under 60% food restriction, AgRP STAT5-knockout mice had a blunted upregulation of hypothalamic Agrp mRNA expression and corticosterone serum levels compared to control mice, suggesting a possible role for STAT5 in AgRP neurons for neuroendocrine adaptations to food restriction. Interestingly, ad libitum fed knockout male mice had reduced Pomc and Ucp-1 mRNA expression compared to control group. Taken together, these results suggest that STAT5 signalling in AgRP neurons regulates body adiposity in female mice, as well as some neuroendocrine adaptations to food restriction.

STAT4 influences the expression of IL-10 in Th17 cells

Abstract Autoimmune diseases such as Multiple Sclerosis (MS), Rheumatoid Arthritis (RA) and Crohn’s Disease (CD) affect more than twenty million people in the United States. While the exact etiology of most autoimmune diseases is unknown, single nucleotide polymorphisms in the STAT4 locus are associated with an elevated risk of developing various autoimmune diseases. Specifically, in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), STAT4 is essential for the development of neuroinflammation. Interestingly, STAT4 is a transcription factor known to be important for Th1 cell differentiation, however we and others have demonstrated that many Th1 associated molecules such as IL-12, IFNγ and Tbet are dispensable for EAE. These data lead us to postulate that STAT4 functions in another effector CD4 T cell, Th17 cells, to drive neuroinflammation. Using an adoptive transfer model, we find that STAT4 is required in a Th17-mediated EAE. Transcriptional profiling of the adoptively transferred Th17 cells reveals that STAT4 influences the expression of over 200 genes, one of them being the immunosuppressive cytokine IL-10. Strikingly, we find an indirect relationship between STAT4 and IL-10 in Th17 cells as well as T regulatory 1 (Tr1) cells. We hypothesize that STAT4 regulates the balance between Th17 and Tr1 cells in the context of autoimmune inflammation. Together, these data illustrate a novel role for STAT4 in controlling the functional and plastic properties of Th17 cells, and this may provide a promising therapeutic target for patients affected by Th17 driven autoimmune diseases.

A10 FOOD ANTIGEN-STRESS INTERACTION LEADS TO INCREASE PAIN SIGNALING IN ILEUM AND COLON VIA STAT6 IN AN IBS MODEL

Abstract Background Abdominal pain can be triggered by food ingestion in IBS patients. Previously we have shown that a food antigen induces local release of immune mediators in the colon that increase dorsal root ganglion (DRG) neuron excitability when there is previous antigen exposure in the presence of psychological stress. However, it is unknown if this effect is limited to the colon. Furthermore, the involvement of histamine in the neuronal hyperexcitability suggests that the stress-food antigen interaction evokes a Th2 immune response. Thus, we sought to investigate the role of STAT6, a transcription factor downstream of Th2 cytokines and important for IgE production. Aims 1) Determine if stress-food antigen interaction leads to release of mediators within the small intestine that increase DRG neuron excitability. 2) Determine the involvement of STAT6 on neuronal hyperexcitability induced by the stress-food antigen interaction. Methods BALB/c mice were exposed to water avoidance stress (WAS) or sham stress (SHAM) for 1 hr daily for 10 days. On day 2–10, mice were exposed to ovalbumin (OVA) or saline (SAL). Seven days later, mice were re-exposed to either OVA or SAL every 2 days for 2 weeks yielding 3 groups: WAS/OVA+OVA, WAS/SAL+OVA, and SHAM/OVA+OVA. STAT6 deficient mice were also exposed to WAS/OVA+OVA protocol. Ileum or colonic supernatants were obtained 4 hours after tissue collection. DRG neurons were incubated overnight with supernatants prior to perforated patch clamp recordings. Neuronal excitability was evaluated by measuring the rheobase (minimum current to elicit an action potential, decreased rheobase indicates increased excitability). Mechanosensitivity of extrinsic afferent nerves innervating distal ileum was examined using ex vivo extracellular afferent nerve recordings. Data was analyzed by one or two-way ANOVA with Bonferroni post-hoc test. Results Ileum supernatants from WAS/OVA+OVA mice increased DRG neuron excitability compared to WAS/SAL+OVA and SHAM/OVA+OVA mice (63.3 ± 6.2 pA vs 83.2 ± 5.4 pA, 86.7 ± 4.5 pA, p<0.05). Ileum afferent nerve response to distention was significantly augmented in WAS/OVA+OVA mice compared to WAS/SAL+OVA and SHAM/OVA+OVA (P<0.05, n=4–7). DRG neurons incubated with WAS/OVA+OVA supernatant from STAT6 deficient mice were less excitable compared to neurons incubated with colonic supernatants from wild type mice (86.5 ± 4.1 pA vs 67.6 ± 4.8 pA, p<0.05). Conclusions Stress-food antigen interaction releases mediators in both the small intestine and colon to increase nociceptive signaling, an important finding as IBS can involve both areas. The release of excitatory mediators within the gut appears to involve STAT6. Thus, a stress-food antigen interaction evoking a Th2 immune response in the gut may be a mechanism underlying food induced symptoms in IBS. Funding Agencies Queen’s University, Department of Medicine

Deciphering key regulators involved in epilepsy-induced cardiac damage through whole transcriptome and proteome analysis in a rat model.

Sudden unexpected death in epilepsy (SUDEP) is a major outcome of cardiac dysfunction in patients with epilepsy. In continuation of our previous work, the present study was envisaged to explore the key regulators responsible for cardiac damage associated with chronic seizures using whole transcriptome and proteome analysis in a rat model of temporal lobe epilepsy. A standard lithium-pilocarpine protocol was used to induce recurrent seizures in rats. The isolated rat heart tissue was subjected to transcriptomic and proteomic analysis. An integrated approach of RNA-Seq, proteomics, and system biology analysis was used to identify key regulators involved in seizure-linked cardiac changes. The analyzed differential expression patterns and network interactions were supported by gene and protein expression studies. Altogether, 1157 differentially expressed genes and 1264 proteins were identified in the cardiac tissue of epileptic animals through RNA-Seq and liquid chromatography with tandem mass spectrometry-based proteomic analysis, respectively. The network analysis revealed seven critical genes-STAT3, Myc, Fos, Erbb2, Erbb3, Notch1,andMapk8-that could play a role in seizure-mediated cardiac changes. The LC-MS/MS analysis supported the activation of the transforming growth factor β (TGF-β) pathway in the heart of epileptic animals. Furthermore, our gene and protein expression studies established a key role ofSTAT3, Erbb,andMapk8to develop cardiac changes linked with recurrent seizures. The present multi-omics study identifiedSTAT3, Mapk8,andErbbas key regulatorsinvolved in seizure-associated cardiac changes. It provided a deeper understanding of molecular, cellular, and network-level operations of the identified regulators that lead to cardiac changes in epilepsy.

Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4+ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC50 of 17.38 versus 22.01 μM for cucurbitacin E and 13.36 versus 24.47 μM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC50 of 9.98 and 29.15 μM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.

STAT6 correlates with response to immune checkpoint blockade therapy and predicts worse survival in thyroid cancer.

Aim: The signal transducer and activator of transcription (STAT) family has been documented. However, the role of STATs in thyroid cancer was not fully studied. Materials & methods: A survival analysis of STATs was performed. The function modulated by STAT6 was examined. The role of STAT6 in cancer immune infiltrates and immune checkpoint blockademolecules was investigated. Results: Only low STAT6 expression correlated with worse survival. STAT6 is involved in cell cycle, cell adhesion, apoptosis and notch signaling pathways. STAT6 was significantly positively associated with immune infiltration of B cells, CD4+ T cells, neutrophils, macrophages, dendritic cellsand the immune checkpoint blockade molecules programmed cell death-ligand 1, programmed cell death-ligand 2and cytotoxic T-lymphocyte-associated protein 4. Conclusion: STAT6 may act as a prognostic biomarker and provide useful information for immunotherapy in thyroid carcinoma.

Stat5a promotes brown adipocyte differentiation and thermogenic program through binding and transactivating the Kdm6a promoter

ABSTRACT Previous studies reported that Stat5 promotes adipogenesis and white adipocyte differentiation. However, the role of Stat5 in brown adipocyte development is poorly understood. We found Stat5a was higher expressed in brown adipocytes than in white adipocytes, and its level was increased during the process of brown adipocyte differentiation. In addition, Stat5a expression was affected by cold stress and high-fat diet-feeding, suggesting a potential role in thermogenesis. Knockdown of Stat5a induced downregulation of brown fat specific genes (UCP1, PGC-1α, Acox-1 and Cidea), while overexpression of Stat5a did the opposite effect. What is more, bioinformatics analysis, ChIP assay and Luciferase activity assay all verified that Stat5a directly bind and transactivate Kdm6a promoter (Lysine-specific demethylase 6A). Further, we found that Stat5a overexpression promoted the expression of Kdm6a and inhibited the trimethylation of H3K27. While inhibiting of Kdm6a reversed the promoting effect of Stat5a overexpression on the expression of brown fat specific genes. Therefore, we conclude that Stat5a participated in brown adipocyte differentiation and thermogenic program through binding and transactivating the Kdm6a promoter. Abbreviations: Stat5: Signal transducers and activators of transcription 5; BAT: brown adipose tissue; WAT; white adipose tissue; eWAT: epididymal white adipose tissue; sWAT: subcutaneous white adipose tissue; SVFs: stromal vascular fractions; UCP1: Uncoupling protein 1; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Acox-1: Peroxisomal acyl-coenzyme A oxidase 1; Cidea: Cell death activator CIDE-A; ChIP: Chromatin Immunoprecipitation; HFD: High fat diet; FBS: Fetal bovine serum; siStat5a: Stat5a siRNA; siKdm6: Kdm6a siRNA; pcDNA-Stat5a: over expression of Stat5a pcDNA3.1 vector; IgG: mouse immunoglobulin G; Kdm6a: Lysine-specific demethylase 6A; H3K27me3: trimethylated H3K27

STAT5 ablation in AgRP neurons increases female adiposity and blunts food restriction adaptations.

AgRP neurons are important players in the control of energy homeostasis and are responsive to several hormones. In addition, STAT5 signalling in the brain, which is activated by metabolic hormones and growth factors, modulates food intake, body fat and glucose homeostasis. Given that, and the absence of studies that describe STAT5 function in AgRP cells, the present study investigated the metabolic effects of Stat5a/b gene ablation in these neurons. We observed that STAT5 signalling in AgRP neurons regulates body fat in female mice. However, male and female STAT5-knockout mice did not exhibit altered food intake, energy expenditure or glucose homeostasis compared to control mice. The counter-regulatory response or glucoprivic hyperphagia induced by 2-deoxy-d-glucose treatment were also not affected by AgRP-specific STAT5 ablation. However, under 60% food restriction, AgRP STAT5-knockout mice had a blunted upregulation of hypothalamic Agrp mRNA expression and corticosterone serum levels compared to control mice, suggesting a possible role for STAT5 in AgRP neurons for neuroendocrine adaptations to food restriction. Interestingly, ad libitum fed knockout male mice had reduced Pomc and Ucp-1 mRNA expression compared to control group. Taken together, these results suggest that STAT5 signalling in AgRP neurons regulates body adiposity in female mice, as well as some neuroendocrine adaptations to food restriction.

A Single Point Mutation in the Mumps V Protein Alters Targeting of the Cellular STAT Pathways Resulting in Virus Attenuation.

Mumps virus (MuV) is a neurotropic non-segmented, negative-stranded, enveloped RNA virus in the Paramyxovirus family. The 15.4 kb genome encodes seven genes, including the V/P, which encodes, among other proteins, the V protein. The MuV V protein has been shown to target the cellular signal transducer and activator of transcription proteins STAT1 and STAT3 for proteasome-mediated degradation. While MuV V protein targeting of STAT1 is generally accepted as a means of limiting innate antiviral responses, the consequence of V protein targeting of STAT3 is less clear. Further, since the MuV V protein targets both STAT1 and STAT3, specifically investigating viral antagonism of STAT3 targeting is challenging. However, a previous study reported that a single amino acid substitution in the MuV V protein (E95D) inhibits targeting of STAT3, but not STAT1. This provided us with a unique opportunity to examine the specific role of STAT 3 in MuV virulence in an in vivo model. Here, using a clone of a wild type MuV strain expressing the E95D mutant V protein, we present data linking inhibition of STAT3 targeting with the accelerated clearance of the virus and reduced neurovirulence in vivo, suggesting its role in promoting antiviral responses. These data suggest a rational approach to virus attenuation that could be exploited for future vaccine development.

Protective effect of silencing Stat1 on high glucose-induced podocytes injury via Forkhead transcription factor O1-regulated the oxidative stress response

BackgroundPodocyte plays an important role in maintaining the integrity and function of the glomerular filtration barrier. Various studies reported that forkhead transcription factor (Fox) O1 played a key role in anti-oxidative signaling. This study aimed to investigate the role of Stat1 in high glucose (HG) -induced podocyte injury.MethodsUnder normal glucose, hypertonic and HG stimulated podocyte conditions, cell counting kit-8 (CCK-8) assay, flow cytometry and western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were respectively carried out to determine cell viability, apoptosis, reactive oxygen species (ROS) production and related genes expressions. We then respectively used silent Stat1, simultaneous silencing Stat1 and FoxO1 and over-expression of FoxO1, to observe whether they/it could reverse the damage of podocytes induced by HG.ResultsHigh glucose attenuated cell survival and promoted cell apoptosis in MPC-5 cells at the same time, and it was also observed to promote the protein expression of Stat1 and the FoxO1 expression inhibition. Silencing Stat1 could reverse HG-induced podocytes injury. Specifically, siStat1 increased cell viability, inhibited cell apoptosis and attenuated ROS level in a high-glucose environment. Cleaved caspase-3 and pro-apoptosis protein Bax was significantly down-regulated, and anti-apoptosis protein Bcl-2 was up-regulated by siStat1. The antioxidant genes Catalase, MnSOD, NQO1 and HO1 were up-regulated by siStat1. We found that silencing FoxO1 reversed the protective effect of siStat1 on the HG-induced podocytes injury.ConclusionsSilencing Stat1 could reverse the effects of high glucose-triggered low cell viability, cell apoptosis and ROS release and the functions of Stat1 might be involved in FoxO1 mediated-oxidative stress in nucleus.

Abstract 202: STAT1 regulates B7H3 mediated angiogenesis in medulloblastoma

Abstract B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, plays a key role in the repression of the immune response by cancer cells. B7-H3 is overexpressed on tumor and tumor-associated cells, making it an interesting therapeutic target. B7-H3 also appears to play a role outside of immune evasion, contributing to the progression of cancer via invasion/migration, angiogenesis, and metastasis. Our previous data has confirmed the presence of B7-H3 in highly aggressive Myc+ medulloblastoma (MB) tumors. Data-mining studies revealed that B7-H3 is correlated with Myc expression in Group 3 and 4 MBs, and is associated with worse patient outcomes. miR-29, a tumor suppressor, has been shown downregulating the expression of B7-H3 in various cancers. We have shown that miR-29 can reduce the angiogenesis abilities of D283 and D425 cells by downregulating B7-H3 in vitro. Subsequent RNA-sequencing analysis of miR-29-transfected D283 cells revealed upregulation of STAT1 activity. Immunoblot analysis and real-time PCR of D283 and D425 cells demonstrated increased expression of p-STAT1, when transfected with miR-29 plasmid. Treatment with all-trans retinoic acid induced p-STAT1 expression, with a concomitant decrease in the expression of B7-H3 and MMP-9. This preliminary data revealed a novel role for STAT1 in potentially regulating B7-H3, and consequently, angiogenesis and metastasis in MB. To date, the role of STAT1 in MB progression has not been fully investigated, and so our results provide a new avenue for developing potential treatments for aggressive MB tumors. Citation Format: Ian J. Purvis, Janardhan Avilala, Maheedhara R. Guda, Sujatha Venkataraman, Rajeev Vibhakar, Andrew J. Tsung, Kiran K. Velpula, Swapna Asuthkar. STAT1 regulates B7H3 mediated angiogenesis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 202.

Abstract 3193: Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses

Abstract Adoptive cell therapy (ACT) of retrovirally transduced (RV) CD8 T cells is a powerful technique that has shown promise in tumor eradication in cancer patients. However, some major barriers to current methods are that ACT is expensive, time consuming, and requires harmful and toxic adjunct procedures. Our laboratory has demonstrated the use of TriVax, a potent peptide vaccination strategy that dramatically expands ACT cell populations and bypasses the necessity for adjunct procedures. The purpose of this project was to enhance current methods of ACT+TriVax by testing an antigen-specific antitumor response of RV CD8 T cells and if it could be improved with constitutively active STAT5 (CA-STAT5) expression, a protein activated downstream several cytokine pathways that have been shown to play a role in increasing CD8 T cell persistence and resistance to apoptosis. We tested the hypothesis that CA-STAT5 in CD8 T cells enhances an antitumor effect by increasing T cell persistence and efficacy. Our results show that TriVax administration selectively expanded frequencies of the ACT cell population expressing gp100-TCR in both blood and spleen. When co-transduced with CA-STAT5, an even higher fold expansion of antigen-specific cells was observed. +CA-STAT5 T cells were able to expand more robustly than -CA-STAT5 T cells upon repeated antigen stimulation (vaccine boost), demonstrating nearly 4000-fold increases in antigen-specific CD8 T cells. +CA-STAT5 T cells also seemed to persist longer in vivo over time, and they expressed lower levels of surface PD-1. Using B16F10 melanoma, ACT+TriVax of these cell populations into tumor-bearing mice demonstrated a powerful antitumor effect, leading to tumor regression in treated groups. CA-STAT5 seemed to recapitulate similar antitumor effects our laboratory observed previously with combinatorial anti-PD-L1 treatment or IL2/anti-IL2 mAb complexes (IL2Cx), suggesting a potential role for STAT5 in resisting the PD-1/PD-L1 inhibitory pathway. When compared to tumor-bearing mice treated with ACT+TriVax with anti-PD-L1 antibody, our approach using +CA-STAT5 T cells generated a comparable antitumor response. Altogether, these results demonstrate that RV CD8 T cells expressing gp100-TCR and CA-STAT5 are capable of antigen-dependent expansion in response to TriVax. CA-STAT5 plays a role in increasing T cell proliferation and persistence, as well as increasing efficacy through mechanisms that seem to indicate resistance to PD-1/PD-L1 inhibition. Citation Format: Aaron E. Fan, Hussein Sultan, Takumi Kumai, Esteban Celis. Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3193.

PS986 SYK‐DEPENDENT P‐STAT5 ACTIVITY IS REQUIRED TO MAINTAIN CLONOGENIC POTENTIAL AND OXPHOS METABOLISM IN ACUTE MYELOID LEUKEMIA (AML) CELLS

Background:SYK kinase is an essential component of integrin b3 signaling, which maintain leukemic stem cell (LSC) transcriptional programs in AML. Genetic or pharmacologic inhibition of SYK leads to increased differentiation, reduced proliferation and apoptosis. However, the comprehensive description of mediators and effectors of SYK signaling in AML and its association with LSC maintenance is lacking.Aims:To characterize the role and identify key downstream mediators of SYK signaling in AML responsible for leukemia survival and LSC maintenance.Methods:AML cells (KG1, MOLM14, TEX) or primary AML blasts were incubated with #R406 SYK inhibitor or with vehicle for 24 h. Activity of SYK, ERK, STAT5 was assessed by western blot and/or intracellular phospho‐flow. Proliferation, apoptosis, clonogenic potential were assessed by MTS, PI staining and methylcellulose colony‐forming assay. Differentiation was assessed by Giemsa and CD14/CD15 staining, qNBT and qPCR. Constitutively active STAT5A1∗6 was electroporated into KG1. ROS level was assessed by DCF staining, mitochondrial mass was assessed by MitoTracker Green FM staining or by mitochondrial DNA content. Expression of MYC, genes involved in mitochondrial biogenesis (TFAM, NRF1, NRF2, EF‐Tu) and encoded by mitochondrial genome (ATP6, ND6) were assessed by western blot or qPCR. The oxygen consumption rate (OCR) was measured using Seahorse 96XF Analyzer.Results:To identify downstream mediators of SYK in AML, we assessed activity of SYK‐dependent signaling molecules in TEX, KG1 and MOLM14 cells. AML cells with active SYK kinase (pSYK) exhibited increased basal level of pERK and pSTAT5. #R406 effectively decreased phosphorylation of these proteins, reduced proliferation, clonogenic potential and induced differentiation and apoptosis. Given the role of STAT5 in self‐renewal of HSC, we assessed the contribution of STAT5 to maintenance of AML cell clonogenic potential. #R406 reduced clonogenicity of control cells, whereas in cells expressing constitutively active STAT5A1∗6, the clonogenic potential was maintained. To test whether inhibition of SYK‐STAT5 axis targets LSCs, we assessed sensitivity of LSC‐enriched TEX cells to the inhibitor and found that the compound markedly reduced their clonogenic potential and induced apoptosis. Next, we sought to identify STAT5‐dependent mechanism of LSC depletion following #R406 treatment. STAT5A1∗6‐expressing cells exhibited higher mitochondrial mass, higher expression of genes encoded by mitochondrial genome, higher expression of MYC and MYC target genes involved in mitochondrial biogenesis and, importantly, higher oxidative metabolism (OCR) as compared to control cells. LSCs sorted from TEX cells exhibited similar phenotype. In cells with forced expression of constitutively active STAT5 (STAT5A1∗6), incubation with #R406 did not significantly decrease these LSC‐associated markers, whereas mock‐transfected cells exhibited profound decrease in their abundance, indicating that STAT5 is a major effector of SYK inhibition in LSC‐like population. SYK inhibition not only reduced the expression of MYC and mitochondrial biogenesis genes in AML cells lines and primary blasts, but also reduced basal and maximal OCR in TEX, KG1 and MOLM14 cells.Summary/Conclusion:Taken together, we found that SYK inhibition targets LSC and reduces clonogenic potential of AML cells by decreasing STAT5 and MYC activity, and, subsequently, by decreasing mitochondrial biogenesis and oxidative metabolism.This work was supported by NCN#2013/11/N/NZ5/03704