STAT5 Activation Research Articles

STAT5 interferes with PD-1 transcriptional activation and affects CD8+ T-cell sensitivity to PD-1-dependent immunoregulation.

Programmed cell death-1 (PD-1) is a co-inhibitory receptor that dampens immune responses upon interaction with PD-L1 and PD-L2. Although PD-1 expression on T cells is known to be activation-dependent, how cytokines modify its regulation is not fully resolved. Using polyclonal T-cell activation to study cytokine-dependent PD-1 regulation, we found that IL-2 inhibited transcriptional up-regulation of PD-1 despite the promotion of T-cell activation. The IL-2-mediated reduction in PD-1 expression augmented CD8+ T-cell activities against PD-L1-expressing target cells. To study the mechanism of PD-1 reduction, we focused on STAT5 activation in the IL-2 signaling pathway. Bioinformatic analysis suggested a novel conserved PD-1 promoter domain where NFAT and STAT5 can potentially compete with each other for binding. NFAT1 interaction with this domain revealed substantial potency in PD-1 transcription compared to STAT5A, and STAT5A overexpression could quench NFAT1-dependent PD-1 up-regulation in a sequence-specific manner. Chromatin immunoprecipitation analysis of activated T cells showed that IL-2 treatment significantly diminished the binding of NFAT1 and NFAT2 in the hypothesized competition site, while STAT5 binding to the same region was increased. These results raise the possibility that the competition of transcriptional factors might be involved in the fine-tuning of PD-1 expression by cytokines such as IL-2.

Pharmacological Inhibition of STAT6 Ameliorates Myeloid Fibroblast Activation and Alternative Macrophage Polarization in Renal Fibrosis.

A hallmark of chronic kidney disease is renal fibrosis, which can result in progressive loss of kidney function. Currently, there is no effective therapy for renal fibrosis. Therefore, there is an urgent need to identify potential drug targets for renal fibrosis. In this study, we examined the effect of a selective STAT6 inhibitor, AS1517499, on myeloid fibroblast activation, macrophage polarization, and development of renal fibrosis in two experimental murine models. To investigate the effect of STAT6 inhibition on myeloid fibroblast activation, macrophage polarization, and kidney fibrosis, wild-type mice were subjected to unilateral ureteral obstruction or folic acid administration and treated with AS1517499. Mice treated with vehicle were used as control. At the end of experiments, kidneys were harvested for analysis of myeloid fibroblast activation, macrophage polarization, and renal fibrosis and function. Unilateral ureteral obstruction or folic acid administration induced STAT6 activation in interstitial cells of the kidney, which was significantly abolished by AS1517499 treatment. Mice treated with AS1517499 accumulated fewer myeloid fibroblasts and myofibroblasts in the kidney with ureteral obstruction or folic acid nephropathy compared with vehicle-treated mice. Moreover, AS1517499 significantly suppressed M2 macrophage polarization in the injured kidney. Furthermore, AS1517499 markedly reduced the expression levels of extracellular matrix proteins, and development of kidney fibrosis and dysfunction. These findings suggest that AS1517499 inhibits STAT6 activation, suppresses myeloid fibroblast activation, reduces M2 macrophage polarization, attenuates extracellular matrix protein production, and preserves kidney function. Therefore, targeting STAT6 with AS1517499 is a novel therapeutic approach for chronic kidney disease.

Robust innate immune responses at the placenta during early gestation may limit in utero HIV transmission.

In 2019, >90% of new HIV infections in infants globally occurred vertically. Studies suggest intrauterine transmission most often occurs in the third trimester; however, there are no mechanistic studies to support these observations. We therefore obtained early/mid-gestation and term placentae from 20 HIV/Hepatitis B/CMV negative women. Isolated primary placental macrophages (Hofbauer cells [HCs]) were exposed to HIV-1BaL and/or interferon (IFN)-α, IFN-β, IFN-λ1, and RIG-I-like receptor (RLR) agonists. qRT-PCR, FACS, ELISA, Luminex, and Western blot analyses determined expression of activation markers, co-receptors, viral antigen, cytokines, antiviral genes, and host proteins. Early gestation HCs express higher levels of CCR5 and exhibit a more activated phenotype. Despite downregulation of CCR5, term HCs were more susceptible to HIV replication. Early gestation HCs displayed a more activated phenotype than term HCs and HIV exposure lead to the further up-regulation of T-cell co-stimulatory and MHC molecules. Limited HIV replication in early/mid gestation HCs was associated with increased secretion of anti-inflammatory cytokines, chemokines, and a more robust antiviral immune response. In contrast, term HCs were more susceptible to HIV replication, associated with dampening of IFN-induced STAT1 and STAT2 protein activation. Treatment of early/mid gestation and term HCs, with type I IFNs or RLR agonists reduced HIV replication, underscoring the importance of IFN and RLR signaling in inducing an antiviral state. Viral recognition and antiviral immunity in early gestation HCs may prevent in utero HIV infection, whereas diminished antiviral responses at term can facilitate transmission. Defining mechanisms and specific timing of vertical transmission are critical for the development of specific vaccines and antiviral therapeutics to prevent new HIV infections in children globally.

Kahweol Exerts Skin Moisturizing Activities by Upregulating STAT1 Activity.

Kahweol is a diterpene present in coffee. Until now, several studies have shown that kahweol has anti-inflammatory and anti-angiogenic functions. Due to the limited research available about skin protection, this study aims to discern the potential abilities of kahweol and the possible regulation targets. First, the cytotoxicity of kahweol was checked by 3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay, while 2,20-azino-bis (3ethylbenzothiazoline-6-sulphonic acid) diammonium salt and 1-diphenyl-2-picryl-hydrazyl were used to examine the radical scavenging ability. Polymerase chain reaction analysis was performed to explore the proper time points and doses affecting skin hydration and barrier-related genes. Luciferase assay and Western blotting were used to explore the possible transcription factors. Finally, fludarabine (a STAT1 inhibitor) was chosen to discern the relationship between skin-moisturizing factors and STAT1. We found that HaCaT cells experienced no toxicity from kahweol, and kahweol displayed moderate radical scavenging ability. Moreover, kahweol increased the outcome of HAS1, HAS2, occludin, and TGM-1 from six hours in a dose-dependent manner as well as the activation of STAT1 from six hours. Additionally, kahweol recovered the suppression of HAS2, STAT1-mediated luciferase activity, and HA secretion, which was all downregulated by fludarabine. In this study, we demonstrated that kahweol promotes skin-moisturizing activities by upregulating STAT1.

Atorvastatin Attenuates Programmed Death Ligand-1 (PD-L1) Induction in Human Hepatocellular Carcinoma Cells.

Liver cancer is the sixth most common cancer worldwide with high morbidity and mortality. Programmed death ligand 1 (PD-L1) is a major ligand of programmed death 1 receptor (PD1), and PD1/PD-L1 checkpoint acts as a negative regulator of the immune system. Cancers evade the host’s immune defense via PD-L1 expression. This study aimed to investigate the effects of tumor-related cytokines, interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) on PD-L1 expression in human hepatocellular carcinoma cells, HepG2. Furthermore, as atorvastatin, a cholesterol-lowering agent, is documented for its immunomodulatory properties, its effect on PD-L1 expression was investigated. In this study, through real-time RT-PCR, Western blot, and immunocytochemistry methods, PD-L1 expression in both mRNA and protein levels was found to be synergistically upregulated in HepG2 by a combination of IFNγ and TNFα, and STAT1 activation was mainly responsible for that synergistic effect. Next, atorvastatin can inhibit the induction of PD-L1 by either IFNγ alone or IFNγ/TNFα combination treatment in HepG2 cells. In conclusion, in HepG2 cells, expression of PD-L1 was augmented by cytokines in the tumor microenvironment, and the effect of atorvastatin on tumor immune response through inhibition of PD-L1 induction should be taken into consideration in cancer patients who have been prescribed atorvastatin.

Non-hematopoietic IL-4R\u03b1 expression contributes to fructose-driven obesity and metabolic sequelae

ObjectiveThe risks of excess sugar intake in addition to high-fat diet consumption on immunopathogenesis of obesity-associated metabolic diseases are poorly defined. Interleukin-4 (IL-4) and IL-13 signaling via IL-4Rα regulates adipose tissue lipolysis, insulin sensitivity, and liver fibrosis in obesity. However, the contribution of IL-4Rα to sugar rich diet-driven obesity and metabolic sequelae remains unknown.MethodsWT, IL-4Rα-deficient (IL-4Rα−/−) and STAT6-deficient mice (STAT6−/−) male mice were fed low-fat chow, high fat (HF) or HF plus high carbohydrate (HC/fructose) diet (HF + HC). Analysis included quantification of: (i) body weight, adiposity, energy expenditure, fructose metabolism, fatty acid oxidation/synthesis, glucose dysmetabolism and hepatocellular damage; (ii) the contribution of the hematopoietic or non-hematopoietic IL-4Rα expression; and (iii) the relevance of IL-4Rα downstream canonical STAT6 signaling pathway in this setting.ResultsWe show that IL-4Rα regulated HF + HC diet-driven weight gain, whole body adiposity, adipose tissue inflammatory gene expression, energy expenditure, locomotor activity, glucose metabolism, hepatic steatosis, hepatic inflammatory gene expression and hepatocellular damage. These effects were potentially, and in part, dependent on non-hematopoietic IL-4Rα expression but were independent of direct STAT6 activation. Mechanistically, hepatic ketohexokinase-A and C expression was dependent on IL-4Rα, as it was reduced in IL-4Rα-deficient mice. KHK activity was also affected by HF + HC dietary challenge. Further, reduced expression/activity of KHK in IL-4Rα mice had a significant effect on fatty acid oxidation and fatty acid synthesis pathways.ConclusionOur findings highlight potential contribution of non-hematopoietic IL-4Rα activation of a non-canonical signaling pathway that regulates the HF + HC diet-driven induction of obesity and severity of obesity-associated sequelae.

Induction of CIITA by IFN-γ in macrophages involves STAT1 activation by JAK and JNK

The induction of major histocompatibility complex (MHC) class II proteins by interferon gamma (IFN-γ) in macrophages play an important role during immune responses. Here we explore the signaling pathways involved in the induction by IFN-γ of the MHC II transactivator (CIIta) required for MHC II transcriptional activation. Cyclophilin A (CypA) is required for IFN-γ-dependent induction of MHC II in macrophages, but not when it is mediated by GM-CSF. The effect of CypA appears to be specific because it does not affect the expression of other molecules or genes triggered by IFN-γ, such as FcγR, NOS2, Lmp2, and Tap1. We found that CypA inhibition blocked the IFN-γ-induced expression of CIIta at the transcriptional level in two phases. In an early phase, during the first 2 h of IFN-γ treatment, STAT1 is phosphorylated at Tyrosine 701 and Serine 727, residues required for the induction of the transcription factor IRF1. In a later phase, STAT1 phosphorylation and JNK activation are required to trigger CIIta expression. CypA is needed for STAT1 phosphorylation in this last phase and to bind the CIIta promoter. Our findings demonstrate that STAT1 is required in a two-step induction of CIIta, once again highlighting the significance of cross talk between signaling pathways in macrophages.

TYK2 as a therapeutic target in the treatment of autoimmune and inflammatory diseases.

JAKs are intracellular protein tyrosine kinases that, through activation of STATs, are responsible for signal transduction pathways that regulate cellular responses to numerous cytokines, growth factors and hormones in many different cells. JAK-STAT signaling plays a key role in regulating immune function, and cytokines - such as IL-23, IL-12 and type I interferons - are central to the pathogenesis of autoimmune diseases, including psoriasis, inflammatory bowel disease and systemic lupus erythematosus. Herethe authors review the evidence for targeting TYK2 as a more specific approach to treating these conditions. TYK2 inhibitors are clinically effective in autoimmune and inflammatory diseases and may avoid some of the complications reported with nonselective JAK inhibitors.

Dectin-1 Controls TSLP-Induced Th2 Response by Regulating STAT3, STAT6, and p50-RelB Activities in Dendritic Cells.

The epithelium-associated cytokine thymic stromal lymphopoietin (TSLP) can induce OX40L and CCL17 expression by myeloid dendritic cells (mDCs), which contributes to aberrant Th2-type immune responses. Herein, we report that such TSLP-induced Th2-type immune response can be effectively controlled by Dectin-1, a C-type lectin receptor expressed by mDCs. Dectin-1 stimulation induced STAT3 activation and decreased the transcriptional activity of p50-RelB, both of which resulted in reduced OX40L expression on TSLP-activated mDCs. Dectin-1 stimulation also suppressed TSLP-induced STAT6 activation, resulting in decreased expression of the Th2 chemoattractant CCL17. We further demonstrated that Dectin-1 activation was capable of suppressing ragweed allergen (Amb a 1)-specific Th2-type T cell response in allergy patients ex vivo and house dust mite allergen (Der p 1)-specific IgE response in non-human primates in vivo. Collectively, this study provides a molecular explanation of Dectin-1-mediated suppression of Th2-type inflammatory responses and suggests Dectin-1 as a target for controlling Th2-type inflammation.

Abstract NG11: Orthogonal IL-9 receptor signaling reprograms T cells to obviate conditioning chemotherapy before adoptive cell therapy

Abstract NG11: Orthogonal IL-9 receptor signaling reprograms T cells to obviate conditioning chemotherapy before adoptive cell therapy

α1-Acid Glycoprotein Enhances the Immunosuppressive and Protumor Functions of Tumor-Associated Macrophages

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell-derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte-derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. SIGNIFICANCE: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

NPM1c Alters FLT3-D835Y Localization and Signaling Thereby Inducing a Fulminant Myeloproliferative Disorder in Mice

Activating mutations in FLT3 and NPM1 are the most common alterations in AML and frequently coincidental. The NPM1 mutation (NPM1c) dislocates the protein to the cytoplasm due to a disruption of the nuclear localization signal. In FLT3, two types of mutations are present: Tandem duplications of the juxtamembraneous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). In murine models, both NPM1c and FLT3-ITD induce a myeloproliferative disease (MPN), while FLT3-TKD expression alone is not sufficient to cause a myeloid disease in mice. Co-expression of NPM1c and FLT3-ITD rapidly induces an AML in C57Bl/6 mice. In the present study, we investigated the impact of NPM1c co-expression with the TKD mutation FLT3-D835Y and the molecular mechanism underlying the functional dependency of those two proteins.For this purpose, we used a heterozygous conditional Npm1c knockin mouse model, where bone marrow cells were harvested, retrovirally infected with MiG- Flt3-D835Y and injected into lethally irradiated C57Bl/6 wt mice. Lymphatic/leukemic organs of moribund mice were analyzed for their immune phenotype by flow cytometry. To investigate the molecular function and mechanism leading to disease induction by these oncogenes, intracellular flow cytometry as well as immunofluorescence imaging of primary murine cells were performed to analyze signaling and localization differences. In order to examine the clinical relevance, AML patient samples harboring either NPM1c and FLT3-TKD mutations or FLT3-TKD mutation in combination with wildtype NPM1 were analyzed by immunohistochemistry and flow cytometry for intracellular signaling mediators.Flt3-D835Y+ Npm1c+ mice rapidly developed an MPN with a median latency of 33 days, whereas Flt3-D835Y+ Npm1 wt mice did not develop a leukemic disease. To investigate the underlying mechanism leading to MPN induction in Flt3-D835Y+ Npm1c+ mice, we analyzed the activation of signaling transducer and activator of transcription (STAT) pathways in primary splenocytes of moribund mice. Strikingly, we were able to detect a profound activation of STAT5 in murine Flt3-D835Y+ Npm1c+ cells, which could not be seen in the Flt3-D835Y+ Npm1 wt cells. Next we asked for the clinical significance of these differential FLT3-TKD signaling capacities and performed immunohistochemistry stainings as well as intracellular flow cytometry of primary AML patient BM at initial diagnosis. In line with the findings in the murine model, immunohistochemic analyses showed significantly increased pSTAT5 signal in AML samples harboring both NPM1c and FLT3-TKD mutations, when compared to FLT3-TKD+ samples without NPM1 alteration. These results could be confirmed by intracellular pSTAT5 staining of primary human AML blasts, where STAT5 activation was significantly elevated in FLT3-TKD patients harboring an NPM1c mutation.To investigate the molecular mechanism underlying these differential signaling capacities we performed immunofluorescence and flow cytometry analyses of murine leukemic cells and could show a profound localization shift of FLT3-TKD from the cell surface to the endoplasmic reticulum only in cells harboring the NPM1c mutation. In NPM1 wt cells FLT3-TKD could exclusively be detected at the cell surface. These results could be confirmed by immunoblotting analyses, where we detect an underglycosylated, intracelullar FLT3-TKD 130 kDa-form in NPM1c+ cells while it is found as a highly glycosylated, cell-surface bound 150 kDa form in cells lacking NPM1c.In the present study, we could demonstrate that co-expression of Npm1c and Flt3-D835Y induces a rapid-onset MPN in C57Bl/6 mice. Interestingly, the presence of NPM1c also alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, and thereby leads to STAT5 activation by FLT3-D835Y. We could further show that STAT5 activation not only occurs in murine cells but also in AML patients with FLT3-TKD in combination with NPM1c mutation. Thus, our data indicate that the NPM1c mutation shifts the signaling properties of FLT3-TKD towards those of the FLT3-ITD mutation, which may explain its occurrence in myeloid disorders and suggest that STAT5 might be a potential unique therapeutic target in FLT3-mutated AML. DisclosuresNo relevant conflicts of interest to declare.

Phospholipase Cgamma Pathway-Dependent mTOR Signaling Controls IL-7-Mediated Early B Cell Development

Early B cell lymphopoiesis is intricately regulated by signals from various receptors including the pre-B cell receptor (pre-BCR) and the interleukin-7 receptor (IL-7R), but the molecular mechanism underlying the regulation is not fully understood. The Phospholipase Cγ (PLCγ) signaling pathway plays a critical role in antigen receptor-mediated activation of mature B cells, but the role of this pathway in cytokine function and early B cell development is completely unknown. Here we generated inducible PLCγ1/PLCγ2 double-deficient mice and found that the mutant mice exhibited a complete blockage of early B cell development at the pre-pro-B cell stage, similar to IL-7R-deficient but not pre-BCR-deficient mice. Of note, PLCγ1/PLCγ2 double-deficient mice had normal numbers of the HSC-containing Lin-c-Kit+Sca1+ (LSK) cells, myeloid progenitor-containing Lin-c-Kit+Sca1- (LK) cells and myeloid lineage cells compared to wild-type mice. PLCγ1/PLCγ2 double-deficient mice displayed a marked reduction in IL-7-induced rearrangement of the distal VHJ558 family gene segments. In addition, PLCγ1/PLCγ2 double deficiency altered the expression of lymphoid-lineage-affiliated genes in common lymphoid progenitors (CLPs) and the expression of B cell lineage-affiliated genes, IL-7 signaling pathway-regulated genes and cell cycle pathway-related genes in B cell progenitors. Further, PLCγ1/PLCγ2 double-deficient Lin- BM progenitors co-cultured with OP-9 stromal cells plus IL-7 failed to give rise to B cells whereas wild-type control BM progenitors generated B cells. PLCγ1/PLCγ2 double-deficient B progenitors exhibited a decrease in the level of IL-7-induced BrdU uptake compared to the corresponding wild-type control cells. Biochemically, the PLCγ pathway was activated upon IL-7 engagement in Rag1-deficient pro-B cells that lack the pre-BCR. PLCγ1/PLCγ2-double deficiency or inhibition of PLCγ pathway by PLC inhibitors impaired IL-7-induced activation of mTORC1 but not Stat5. Consistently, inhibition of the PLCγ pathway impaired IL-7-mediated metabolism of B cell progenitors. It's known that PLCγ activates the DAG/PKC-dependent and IP3/Ca2+-dependent signaling pathways, the two major downstream signaling cascades. We found that inhibition of the PKC-dependent, but not the Ca2+-dependent, pathway impaired IL-7-mediated B cell production and mTORC1 but not Stat5 activation. In addition, the PKC agonist PDBu induced mTORC1 activation in wild-type B cell progenitors, and stimulated 3H-thymidine incorporation in PLCγ1/PLCγ2 double-deficient B cell progenitors to the same extent as it did in wild-type control cells. Moreover, disruption of conventional Akt/TSC/Rheb signaling axis did not affect IL-7-controlled mTORC1 activation or early B cell development. Taken together, our current study for the first time demonstrates that the PLCγ pathway is critical for cytokine-mediated biological function, such as IL-7R-regulated early B lymphopoiesis, and this pathway directs IL-7R-mediated early B cell development through a novel and thitherto-unknown PLCγ/DAG/PKC-dependent but not conventional Akt/Rheb-dependent activation of mTORC1. DisclosuresNo relevant conflicts of interest to declare.

Clinically used JAK inhibitor blunts dsRNA-induced inflammation and calcification in aortic valve interstitial cells.

Calcific aortic valve disease (CAVD) is the most prevalent valvulopathy worldwide. Growing evidence supports a role for viral and cell-derived double-stranded (ds)-RNA in cardiovascular pathophysiology. Poly(I:C), a dsRNA surrogate, has been shown to induce inflammation, type I interferon (IFN) responses, and osteogenesis through Toll-like receptor 3 in aortic valve interstitial cells (VIC). Here, we aimed to determine whether IFN signaling via Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) mediates dsRNA-induced responses in primary human VIC. Western blot, ELISA, qPCR, calcification, flow cytometry, and enzymatic assays were performed to evaluate the mechanisms of dsRNA-induced inflammation and calcification. Poly(I:C) triggered a type I IFN response characterized by IFN-regulatory factors gene upregulation, IFN-β secretion, and STAT1 activation. Additionally, Poly(I:C) promoted VIC inflammation via NF-κB and subsequent adhesion molecule expression, and cytokine secretion. Pretreatment with ruxolitinib, a clinically used JAK inhibitor, abrogated these responses. Moreover, Poly(I:C) promoted a pro-osteogenic phenotype and increased VIC calcification to a higher extent in cells from males. Inhibition of JAK with ruxolitinib or a type I IFN receptor blocking antibody blunted Poly(I:C)-induced calcification. Mechanistically, Poly(I:C) promoted VIC apoptosis in calcification medium, which was inhibited by ruxolitinib. Moreover, Poly(I:C) co-operated with IFN-γ to increase VIC calcification by synergistically activating extracellular signal-regulated kinases and hypoxia-inducible factor-1α pathways. In conclusion, JAK/STAT signaling mediates dsRNA-triggered inflammation, apoptosis, and calcification and may contribute to a positive autocrine loop in human VIC in the presence of IFN-γ. Blockade of dsRNA responses with JAK inhibitors may be a promising therapeutic avenue for CAVD.

Biologic for the Treatment of Ph-like B-Cell Acute Lymphoblastic Leukemia with Overexpression of CRLF2

Ph-like B-cell acute lymphoblastic leukemia (B-ALL) is associated with poor outcomes and high relapse rates. Approximately 50% of Ph-like B-ALL cases occur due to genetic alterations leading to overexpression of the cytokine receptor, CRLF2. B-ALL with overexpression of CRLF2 (CRLF2 B-ALL) occurs 5 times more often in Hispanic children than others, is prevalent in adolescents and young adults, and makes up ~1/3 of adult B-ALL. CRLF2, together with the IL-7Rα, forms a receptor complex that can be activated by circulating TSLP cytokine in patients. The human CRLF2 receptor is not activated by mouse TSLP so classic patient-derived xenografts (PDX) do not model TSLP-induced CRLF2 signaling that occurs in patients. CRLF2-mediated JAK/STAT and PI3/AKT/mTOR signals are believed to contribute to survival and proliferation of leukemia cells. We developed a novel PDX model of CRLF2 B-ALL that allows us to vary circulating levels of human TSLP (+T PDX). Primary CRLF2 B-ALL cells injected into +T PDX with circulating human TSLP (hTSLP) levels similar to pediatric leukemia patients (~4-10 pg/ml), engrafted well and showed a gene expression pattern that was more similar to the original patient sample than when injected into classic PDX. To our surprise, when +T PDX expressed physiological, but elevated levels of hTSLP (> 40 pg/ml, upper end of range reported in healthy children), CRLF2 B-ALL cells were essentially eliminated, but grew robustly in PDX without hTSLP (-T PDX). These results have been observed in 4 independent experiments for a total of 17 +T PDX and 12 -T PDX mice produced using CRLF2 B-ALL cells from two different Hispanic pediatric patients with CRLF2 B-ALL. We hypothesize that anti-leukemia effects observed at hTSLP concentrations above 40 pg/ml are mediated via TSLP-induced upregulation of the Suppressor of Cytokine Signaling (SOCS) genes. SOCS genes encode a family of proteins that regulate cytokine signaling via negative feedback through multiple mechanisms including ubiquitin-mediated degradation of JAKs and cytokine receptor components. Using the CRLF2 B-ALL cell lines, MUTZ5 and CALL-4, we found that SOCS family proteins encoded by the SOCS1 and SOCS3 genes were upregulated following culture with 15 ng/ml hTSLP, as compared to controls without hTSLP. Similarly, primary CRLF2 B-ALL cells from Hispanic pediatric patients cultured with TSLP showed upregulation of SOCS1 and SOCS3 mRNA and proteins. Next, we determined whether the upregulation of SOCS proteins was accompanied by a loss of CRLF2-mediated signals. We found that CRLF2 B-ALL cell lines and primary cells cultured with hTSLP lost the ability to upregulate STAT5 and S6 phosphorylation following hTSLP stimulation. Further, cells cultured with TSLP showed a loss of cytokine receptors as well as STAT5 phosphorylation levels that were below the baseline observed in leukemia cells never exposed to hTSLP. These data suggest that TSLP exerts anti-leukemia effects by shutting down CRLF2-mediated signals and that this shut down may occur through SOCS-mediated degradation of cytokine receptor components and/or degradation of the mutant JAK2 that provides constitutively low level STAT5 activation in these CRLF2 B-ALL cell lines and in a majority of patients with this disease. In summary, these results provide evidence that elevated hTSLP exerts a therapeutic effect on CRLF2 B-ALL and suggest that this occurs via SOCS-mediated suppression of the CRLF2 signaling pathway. These studies identify the human TSLP cytokine as a potential biologic therapy to treat CRLF2 B-ALL and reduce cancer health disparities for Hispanic children with CRLF2 B-ALL. Supported by 1R01CA209829. DisclosuresCoats:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees. Dovat:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees. Payne:ELF Zone Inc: Membership on an entity's Board of Directors or advisory committees; ELF Zone Inc: Equity Ownership.

NPM1-ALK Overexpression-Driven Toxicity in ALCL Is Partner Dependent and Mediated By STAT1 Antagonism of STAT3 Survival Signaling, Revealing a Novel Therapeutic Strategy

Activation of the tyrosine-kinase domain (TKD) of anaplastic lymphoma kinase (ALK) drives malignant growth in subsets of several cancers as part of oncogenic fusions resulting from chromosomal rearrangements. Clinically, 70% of anaplastic large-cell lymphoma (ALCL) and ~5% of non-small-cell lung cancer (NSCLC) are ALK+. ALK tyrosine-kinase inhibitors (TKIs) are approved for treatment of NSCLC and are under investigation in ALCL. Clinical resistance in lung cancer typically develops within 10-12 months and is due most commonly to activation of alternate pathways or second-site kinase-domain mutations. In contrast, in ALK + ALCL, we and others have shown that over-expression of the NPM1-ALK fusion kinase is the major mechanism of TKI resistance. While this permits cells to survive TKI treatment, it results in a toxic signaling overdose upon inhibitor withdrawal both in vitro and in vivo. Therefore, NPM1-ALK is both essential to the survival of ALK+ ALCL and able to induce apoptosis when activated above a certain threshold. We now report the mechanism underlying NPM1-ALK signaling overdose.We find that all generations of ALK TKIs drive NPM1-ALK overexpression as the most frequent resistance mechanism in ALCL cells, but increased ALK activity upon inhibitor withdrawal is universally toxic. ALK stimulates several oncogenic pathways, including JAK/STAT3, MEK/ERK, and PI3K/AKT, but we find none of these canonical downstream pathways is a key driver of overdose toxicity. Novel analysis of viability data using a normalized weighted area under the curve to quantify level of rescue showed that only ALK inhibitors can rescue from ALK overdose signaling. We also observed that NPM1-ALK over-expression has a significantly more detrimental effect on cell fitness than the lung cancer fusion EML4-ALK, suggesting that the toxicity seen upon overdose is fusion-partner dependent. For confirmation, we generated constitutively active mutants of the ALK TKD with no fusion partner that are potently transforming but do not impair cell fitness upon ALK overdose. Phosphoproteomics analysis of resistant sub-clones during overdose as compared to parental lines in vehicle show an overlap of over 1,700 targets driven by ALK signaling. Furthermore, comparison of the parental line in drug to the resistant line undergoing overdose, 178 genes group together in the overall death state. These analyses revealed 21 phospho targets uniquely upregulated during ALK overdose (p <0.05). Annotation of these genes align with RNA-seq data showing increased phosphorylation of proteins driving the same pathways.STAT1 was a prominent target further pursued as the driver of apoptosis during NPMI-ALK overdose. Normally activated by INF-γ, STAT1 antagonizes the transcriptional output of STAT3, the core survival pathway of ALCL. It previously was shown to be a direct phospho target of NPM1-ALK, which at steady state results in its proteasomal degradation permitting predominance of the STAT3 transcriptional program. Upon NPM-ALK overdose, however, we see rapid and dramatic upregulation of phosphorylated STAT1 (Y705), in both the cytoplasm and nucleus. RNA-Seq analysis at the gene level shows loss of STAT3 targets but up-regulation of the STAT1 program, suggesting that during NPM1-ALK overdose STAT1 activation antagonizes STAT3 survival output required for ALCL cell viability and driving them to apoptosis. IFN-γ, already known to be detrimental to ALK+ ALCL cells as a single agent, combined with ALK inhibitors therefore represents a novel treatment strategy able to synergistically deactivate the core survival pathway of ALK+ ALCL. DisclosuresNo relevant conflicts of interest to declare.

Higher Rates of Molecular Response to Peg-IFNa in JAK2V617F Vs. Calr Mutant MPN Patients Are Due to JAK1-Mediated STAT1 Activation and Autoregulation

Introduction: Pegylated interferon alpha (Peg-IFNa) treatment has been described to induce molecular remission in patients (pts) with myeloproliferative neoplasms (MPN), with partial molecular remission (PMR) being achieved in approximately 30-40% of pts. However, it is unclear whether pts with CALR mutations exhibit the same response as pts with JAK2V617F mutations. Also, the precise mechanisms accounting for the responses are still unknown.Methods: We report on hematological and molecular responses, according to ELN guidelines, in 52 MPN pts (JAK2V617F n=26 vs. CALR mutation n=26; ET n=25, prePMF n=8, PMF n=19), treated at Roskilde hospital with Peg-IFNa. The mechanism of IFNa response was assessed in stably transduced 32D murine cell lines and in a cohort of MPN patients within the Czech MPN registry MIND. All pts provided written informed consent, as approved by the local ethics committees. Cell proliferation was assessed by cell counting and/or MTT assay, signaling molecules were quantified using RT-PCR, flow cytometry and/or Western blotting.Results: Pts harboring JAK2V617F mutations were more likely to achieve PMR than those harboring CALR mutations (60% vs. 23%, p<0.05 by Chi square testing), while there was no significant difference in allelic burden before Peg-IFNa treatment. Accordingly, the median allelic burden decrease from baseline in the IFNa responding pts was 66% (range 15-99%) in JAK2V617F- vs. 29% (range 5-82%) in CALR-mutant pts. These differences were present independently of the fractions of ET, prePMF, or PMF pts in the two groups. There was no significant difference in the rates of hematological response. When examining primary cells from the Czech MPN cohort, cells from JAK2V617F positive pts showed higher expression of IFNa response-related genes such as Irf1 (p=0.0350) and Stat1 (trend at p=0.0874). Moreover, IFNa receptor expression was higher in JAK2V617F positive pts (p=0.0471) and the expression levels were associated with hemoglobin (p=0.0139) and hematocrit levels (p=0.0251). This was reflected in 32D cell lines, ectopically expressing both the MPL receptor and either JAK2V617F or CALR del 52. Here, the expression of Irf7, Irf-9, Ifi-27, and Stat2 was significantly higher in JAK2VF vs. CALR mutants (all p<0.001). In addition, JAK2V617F positive cells showed higher STAT1 phosphorylation and Stat1 mRNA expression levels (p=0.0499). In particular, STAT1 showed constitutive phosphorylation in JAK2V617F but not CALR mutant cells, indicating priming of these cells towards an IFNa response. Overexpression of STAT1 but not of a dominant-negative STAT1YF mutant was able to rescue cellular IFNa-induced growth arrest in CALR positive cells to the same level of JAK2V617F positive cells. In contrast, manipulation of STAT2 expression did not alter IFNa signaling. IFNa-induced growth arrest was counteracted by selective JAK1 inhibition (using itacitinib or filgotinib) but not selective JAK2 inhibition (using BMS-911543).Conclusions: MPN pts harboring JAK2V617F mutations show superior Peg-IFNa sensitivity against the malignant clone, as detected by molecular responses, compared to pts harboring CALR mutations. The higher IFNa sensitivity of JAK2V617F mutant cells was reflected in mutant-specific cell lines and is at least in part due to JAK1-mediated increases in STAT1 phosphorylation and STAT1 transcription. These results are of considerable importance for future clinical trials of Peg-IFNa as monotherapy or in combination with JAK inhibitors. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding. Knudsen:Novartis: Other: Payed for ASH2016 registration fee, travel expenses and accommodation. Hasselbalch:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOR Orphan: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.

SOCS5 Regulates JAK-STAT Signaling and T-ALL Development

T-lineage acute lymphoblastic leukemia (T-ALL) is a hematopoietic disease that tends to present with high-risk features in children, adolescents and young adults. While cure rates have improved in recent decades, event free survival is <10% in patients with relapsed disease. Activating mutations in IL-7, JAK1, JAK2, JAK3 have been implicated in aberrant JAK/STAT signal transduction and disease progression. However, the roles played by suppressors of the cytokine signaling (SOCS) remain incompletely understood. Because SOCS5 plays a key regulatory role in T-helper (Th) cell differentiation, we hypothesized that this gene is a critical determinant in mediating aberrant JAK/STAT signaling in T-ALL. To test regulatory effects of SOCS5 on T-ALL signal transduction and progression, we utilized lentiviral shRNA-mediated methods to knock down of SOCS5 in PF382 and KoptK1 T-ALL cell lines or, alternatively, to force over-expression of SOCS5 in ALL-SIL cells, respectively. We found that SOCS5 silencing resulted in enhanced cellular proliferation (P < 0.05), and that forced expression of SOCS5 resulted in significant decrease in cell proliferation rate (p < 0.05), suggesting that SOCS5 negatively regulates T-ALL cell growth in vitro. We utilized flow-based assays to examine the effects of SOCS5 on cell cycle control, and found that SOCS5 depletion significantly increased cell cycle progression in S/G2/M phases and decreased G1 (p < 0.05), but had no effect on apoptotic cell death. Forced expression of SOCS5 delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of cell cycle (p < 0.05). We next utilized protein analyses to examine the effects of SOCS5 on JAK-STAT signaling and found that suppressed SOCS5 expression induced phosphorylation of STAT1, STAT5, STAT6 while forced SOCS5 expression decreased STATs activation. Because STAT6 is involved in regulation of IL4 signaling we tested whether SOCS5 regulates the expression of this cytokine and its receptor, IL4R and demonstrated that SOCS5 depletion upregulated IL4 and its receptor in CCRF-CEM and KoptK1 cells. We next asked whether SOCS5 regulates IL7 signaling, which is critical for T-ALL development and resistance. Down-regulation of SOCS5 expression enhanced expression of both, IL7 and IL7R. On the contrary, SOCS5 overexpression decreased the levels of IL7 and IL7R. These findings suggest that SOCS5 is involved in negative regulation of IL4 and IL7 signaling. Similar experiments showed the SOCS5 leads to aberrant signaling and expression of c-myc oncogene. To study the effect of SOCS5 on leukemia development in a murine xenograft model, PF382 cells transduced with SOCS5 shRNA and control plasmid were transplanted intravenously into the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (10 mice per group) followed by survival analyses. We observed a significant decrease in mouse survival after SOCS5 depletion (median survival - 21 days) compared to animals engrafted with negative control cells (median survival - 30 days; p < 0.0002), indicating that SOCS5 inactivation promotes T-ALL progression. Using the same murine model, depletion of SOCS5 significantly increased the number of leukemic cells in bone marrow (92.4 ± 3.5 % vs. 13.6 ± 6 %; p < 0.0001), brain (88.4 ± 1.5 % vs. 14.7 ± 5.6 %; p < 0.0001) and liver (49.5 ± 8.2 % vs. 6.1 ± 1.5 %; p < 0.002) compared to negative control group. Our results link the aberrant SOCS5 expression to the up-regulation of the JAK-STAT and cytokine receptor-signaling cascade in T-ALL. We propose deregulation of SOCS5 expression as a novel mechanism governing aberrant cytokine signaling and JAK-STAT signal transduction in T-ALL. We conclude that SOCS5 is an important regulator of T-ALL cell proliferation and leukemic progression in sanctuary and non-sanctuary sites. DisclosuresNo relevant conflicts of interest to declare.

PRL-3 induces a positive signaling circuit between glycolysis and activation of STAT1/2.

Multiple myeloma (MM) is an incurable hematologic malignancy resulting from the clonal expansion of plasma cells. MM cells are interacting with components of the bone marrow microenvironment such as cytokines to survive and proliferate. Phosphatase of regenerating liver (PRL)-3, a cytokine-induced oncogenic phosphatase, is highly expressed in myeloma patients and is a mediator of metabolic reprogramming of cancer cells. To find novel pathways and genes regulated by PRL-3, we characterized the global transcriptional response to PRL-3 overexpression in two MM cell lines. We used pathway enrichment analysis to identify pathways regulated by PRL-3. We further confirmed the hits from the enrichment analysis with in vitro experiments and investigated their function. We found that PRL-3 induced expression of genes belonging to the type 1 interferon (IFN-I) signaling pathway due to activation of signal transducer and activator of transcription (STAT) 1 and STAT2. This activation was independent of autocrine IFN-I secretion. The increase in STAT1 and STAT2 did not result in any of the common consequences of increased IFN-I or STAT1 signaling in cancer. Knockdown of STAT1/2 did not affect the viability of the cells, but decreased PRL-3-induced glycolysis. Interestingly, glucose metabolism contributed to the activation of STAT1 and STAT2 and expression of IFN-I-stimulated genes in PRL-3-overexpressing cells. In summary, we describe a novel signaling circuit where the key IFN-I-activated transcription factors STAT1 and STAT2 are important drivers of the increase in glycolysis induced by PRL-3. Subsequently, increased glycolysis regulates the IFN-I-stimulated genes by augmenting the activation of STAT1/2.

Protectin D1 reduces imiquimod-induced psoriasiform skin inflammation

Specialized proresolving mediators are enzymatically oxygenated natural molecules derived from polyunsaturated fatty acids and are considered novel. These novel mediators include lipoxins from arachidonic acid, resolvins and protectins from omega-3 essential fatty acids, and new maresins. These mediators harbor potent dual proresolving and anti-inflammatory properties. Resolvins and protectins are known to be potent when administered to various inflammation-associated animal models of human diseases. Although psoriasis’ etiology remains unknown, there is accumulating evidence indicating that cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-23, and IL-17, play pivotal roles in its development. Experimentally, resolvins, maresins, and lipoxins downregulate the cytokine expression of the IL-23/IL-17 axis and inhibition of mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) cell signaling transduction pathways. Here, we assessed the effects of protectin D1 (PD1) on imiquimod (IMQ)-induced psoriasiform skin inflammation and keratinocytes. PD1 showed clinical improvement in skin thickness, redness, and scaling in psoriasis mouse models. Moreover, PD1 decreased IL-1β, IL-6, IL-17, and CXCL1 mRNA expressions and reduced STAT1 and NF-κB signaling pathway activation in lesions. Serum myeloperoxidase, IgG2a, IL-1β, IL-6, IL-17, and TNF-α and spleen CD4+IFN-γ+IL-17+ T lymphocytes were reduced after PD1 treatment in IMQ-induced psoriasiform mouse models. In addition, IL-1β, IL-6, IL-8, and IL-18BP gene expressions were decreased in PD1-treated keratinocytes. Moreover, a decrease in the expression levels of CCL17 and IL-6 and an inhibition of the STAT1 and NF-κB signaling transduction pathways was observed in keratinocytes. These PD1 anti-inflammatory effects suggest that it is a good therapeutic candidate for psoriasis.