Phosphorylation Of STAT Proteins Research Articles

A Novel STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition in a Wide Range of Hematopoietic Malignancies without Growth Suppression of Normal Hematopoietic Cells

Abstract 577Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like JAK family kinases and Src family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials.Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases and Src family kinases. In HEL92.1.7 cells with constitutively active mutation of JAK2, OPB-31121 treatment inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). Src-dependent constitutive phosphorylation of STAT3 was also inhibited by OPB-31121 without inhibition of Src in H1650 cells that had active mutantation of EGF receptor. In addition, STAT3 immunoprecipitated from OPB-31121-treated cells was neither phosphorylated by JAK2 nor Lyn, a Src family kinase, in vitro without decrease in auto phosphorylation of upstream kinases,OPB-31121 demonstrated strong growth inhibitory effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including myeloma, AML with JAK2 mutation and CML. It is revealed that STAT3 is constitutively activated by oncogenic autocrine of IL-6 pathway or tyrosine kinase signal from oncoprotein in these cell lines. We also demonstrated growth inhibition or reduction of cell lines including HEL92.1.7 (AML with JAK2 mutation, T/C: 16%), KU812 (CML, T/C: 2%), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation, T/C: 5.9%) in NOD/SCID mice. For further analyses, we used human leukemia model mice where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth reduction of leukemia cells of BCR-ABL-positive ALL (T/C: 4%, Figure B), CML-BC with T315I mutation in BCR-ABL (T/C: 15.3%), and AML (T/C: 15.9%). Notably, OPB-31121-induced growth reduction was extremely selective for leukemia cells. Normal hematopoietic cells of mice were hardly affected by OPB-31121, whereas, cytarabine showed non-specific growth suppression of both leukemia cells and normal hematopoietic cells (Figure C). The safety of OPB-31121 on normal hematopoietic cells was also confirmed by colony formation assay, where OPB-31121 hardly affected colony formation of human cord blood cells at 100 nM. For further analyses, we transplanted human cord blood cells into NOG mice and investigated the growth inhibitory effect of OPB-31121 on normal hematopoietic cells in vivo. No significant growth suppression of human normal hematopoietic cells was observed in OPB-31121 treated mice (T/C: 99.9%, Figure D).Taken together, we conclude that OPB-31121 holds promise as a non-myelosuppressive therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase I/II trial in Japan. [Display omitted] Disclosures:Hayakawa:Otsuka Pharmaceutical Co. Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co. Ltd.: Employment. Sumida:otsuka Pharmaceutical Co. Ltd.: Employment. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

HIV interferes with SOCS‐1 and ‐3 expression levels driving immune activation

HIV infection is characterized by sustained immune activation, which is reflected by activated T cells and, in particular, by increased levels of phosphorylated STAT proteins. Here, we hypothesized that T-cell activation in HIV infection is partially due to the inability of SOCS-1 and SOCS-3 to control the JAK/STAT pathway. We found higher levels of SOCS-1/3 mRNA levels in CD4(+) T cells of HIV-infected patients than in healthy controls. However, SOCS protein levels were lower, explaining the lack of attenuation of the JAK/STAT pathway. Infection of CD4(+) T cells alone did not activate STATs, while ex vivo infection of PBMC did, indicating that non-T cells critical for shaping the immune response, e.g. DC were responsible for the STAT-1 activation. Supernatants from ex vivo-infected PBMC transferred to CD4(+) T cells induced JAK/STAT activation, pointing to a central role of soluble factors. Notably, over-expression of SOCS-1/3 in CD4(+) T cells prevented JAK/STAT activation. Thus, HIV infection interferes with SOCS-1/3 expression driving immune activation. Sustained immune activation disrupts the lymphoid system and favors HIV replication since HIV preferentially infects activated cells. We speculate that regulating SOCS may be a potential way to counteract immune activation in HIV disease.

Mechanisms of STAT Protein Activation by Oncogenic KIT Mutants in Neoplastic Mast Cells

Mutations in the c-kit gene occur in the vast majority of mastocytosis. In adult patients as well as in the cell line derived from mast cell neoplasms, the mutations occur almost exclusively at amino acid 816 within the kinase domain of KIT. Among the downstream effectors of KIT signaling, STAT3 and STAT5 have been shown to be critical for cell proliferation elicited by the KIT-Asp(816) mutant protein. However, little is known about the mechanisms of activation of STAT proteins. In this study, we identify and clarify the contribution of various STAT kinases in two widely used neoplastic mast cell lines, P815 and HMC-1. We show that STAT1, -3, and -5 proteins are activated downstream of the KIT-Asp(816) mutant. All three STAT proteins are located in the nucleus and are phosphorylated on serine residues. KIT-Asp(816) mutant can directly phosphorylate STATs on the activation-specific tyrosine residues in vitro. However, within cells, SRC family kinases and JAKs diversely contribute to tyrosine phosphorylation of STAT proteins downstream of the KIT mutant. Using a panel of inhibitors, we provide evidence for the implication or exclusion of serine/threonine kinases as responsible for serine phosphorylation of STAT1, -3, and -5 in the two cell lines. Finally, we show that only STAT5 is transcriptionally active in these cells. This suggests that the contribution of STAT1 and STAT3 downstream of KIT mutant is independent of their transcription factor function.

Effects of IL-6 on invasion of pancreatic cancer cells and its mechanism

Objective To investigate the effects and mechanism of IL-6 on invasion and metastasis of human pancreatic cancer cells. Methods IL-6 was added into the culture media of human pancreatic cancer cells Capan-2 and SW1990. Cell growth was measured by MTT assay. Western blot and immunocytochemistry were performed to detect Phosphorylated STAT3 (P-STAT3) protein. VEGF and MMP-2 mRNA and protein expression were examined using fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blot, respectively. The invasion ability of SW1990 and Capan2 cells was determined by cell invasion assay in vitro. Results 100 ng/mL IL-6 significantly promoted growth and invasion ability of Capan-2 and SW1990 cells (P＜0.05). The use of IL-6 not only markedly increased the protein expression of P-STAT3, VEGF and MMP-2, but also greatly increased the mRNA expression of MMP-2 and VEGF. Conclusions STAT3 signal transducer pathway activation with IL-6 can promote the invasion ability of pancreatic cancer cells in vitro through up-regulation of MMP-2 and VEGF expression. STAT3 signal transducer may provide a novel therapeutic target for the treatment of pancreatic cancer. Key words: Pancreatic cancer; Interleukin-6; STAT3; Invasion

A Novel Direct STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition In a Wide Range of Hematopoietic Malignancies and Effectively Suppresses the Chemotherapy Resistant Quiescent Cells In Vivo

AbstractAbstract 3277Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like Janus kinase (JAK) family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis-resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials.Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases. OPB-31121 treatment of HEL92.1.7 cells that had constitutive active mutation of JAK2 inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). STAT3 phosphorylation by JAK2 in vitro was also inhibited by OPB-31121 under constant JAK2 autophosphorylation. On the other hand, it did not inhibit dimerization and nuclear translocation of STAT3 once STAT3 was phosphorylated. Also, direct association between OPB-31121 and STAT3 was suggested in vitro. These data implies that one of the mechanisms of OPB-31121 action was the direct inhibition of STAT3 phosphorylation without JAK kinase inhibition. [Display omitted] OPB-31121 demonstrated strong growth suppressive effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including acute myeloid leukemia (AML) with JAK2 mutation or fms-related tyrosine kinase 3 (FLT3) mutation, chronic myeloid leukemia (CML), and myeloma. It is revealed that STAT3 is constitutively activated by tyrosine kinase signal from oncoprotein or oncogenic autocrine of IL-6 pathway in these cell lines. Of note, OPB-31121 had little growth inhibitory effect on normal hematopoietic cells and hardly affected colony formation of human cord blood cells at 100 nM. We also demonstrated growth suppression or regression of cell lines including HEL92.1.7, KU812 (CML), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation) in NOD/SCID mice (T/C: 1.8 to 39.5 %). For further analyses, we used human leukemia model mouse where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth suppression of leukemia cells of BCR-ABL-positive acute lymphoblastic leukemia (ALL), CML-blast crisis (BC), CML-BC with T315I mutation in BCR-ABL, and AML with FLT3/ITD (T/C: 4 to 58 %, Figure B). Furthermore, treatment with cytarabine induced accumulation of quiescent cells that were thought to be relatively resistant to chemotherapy, whereas OPB-31121 did not cause such accumulation, suggesting its effectiveness on quiescent cells (Figure C). We are now investigating the effect of OPB-31121 on leukemia-initiating cells and the results will be shown at the meeting. Taken together, we conclude that OPB-31121 holds promise as a therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase 1 trial in Hong-Kong, Korea, and the USA. Disclosures:Hayakawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Matsuyama:Otsuka Pharmaceutical Co., Ltd.: Employment. Harada:Otsuka Pharmaceutical Co., Ltd.: Employment. Hashimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Ohi:Otsuka Pharmaceutical Co., Ltd.: Employment. Kodama:Otsuka Pharmaceutical Co., Ltd.: Employment. Sumida:Otsuka Pharmaceutical Co., Ltd.: Employment. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.

Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3

Brain microvascular endothelial cells are part of the blood–brain barrier and participate actively in immunological processes including cytokine-mediated inflammatory reactions. Using the human brain capillary endothelial cell line hCMEC/D3, activation of JAK/STAT signaling pathways were studied in response to stimulation by cytokines. The phenotype of hCMEC/D3 cells was confirmed by flow cytometry analysis of cell adhesion factors (cluster of differentiation molecules CD31 and CD34) and the von Willebrand factor endothelial marker was detected by immunofluorescence. Strong STAT1, STAT6 and STAT3 activation was observed in response to interferon-gamma (IFN-γ), interleukin 4 (IL-4) and interleukin 6 (IL-6), respectively. Nuclear translocation of phosphorylated STAT proteins was visualized by confocal microscopy. Treatment of hCMEC/D3 cells with IFN-γ resulted in interferon-induced upregulation of major histocompatibility complex (MHC) class I within 48 h. Interferon-alpha (IFN-α) did not activate STAT1 or STAT3 nor did it induce MHC class I upregulation. Therefore, hCMEC/D3 cells were judged to be non-responsive to IFN-α. We also observed that hCMEC/D3 cells exhibit functional expression of alternative cytokine signal transduction pathways (i.e. TNF-α mediated activation of NF-κB). Together these results indicate that human blood–brain barrier hCMEC/D3 cells are responsive towards stimulation with various cytokines. We conclude that this unique cell line can be used to explore in vitro human blood–brain barrier functionality under proinflammatory conditions.

Upregulation of SOX2, NOTCH1, and ID1 in supratentorial primitive neuroectodermal tumors: a distinct differentiation pattern from that of medulloblastomas

Supratentorial primitive neuroectodermal tumor (PNET) and medulloblastoma are highly malignant embryonal brain tumors. They share morphological similarities, but differ in their differentiation patterns and global gene expression. The authors compared the expression of specific genes involved in neuroglial differentiation in supratentorial PNETs and medulloblastomas to define the distinct characters of these tumors. The mRNA expression of 8 genes (SOX2, NOTCH1, ID1, ASCL-1, NEUROD1, NEUROG1, NEUROG2, and NRG1) was evaluated in 25 embryonal tumors (12 supratentorial PNETs and 13 medulloblastomas) by quantitative real-time polymerase chain reaction. The expression levels of the transcripts of these genes were compared between the tumor groups. Activation of the JAK/STAT3 pathway was assessed by immunoblotting. Relative expression levels of STAT3 and phosphorylated STAT3 proteins were compared. Supratentorial PNETs expressed significantly higher levels of SOX2, NOTCH1, ID1, and ASCL-1 transcripts, whereas the transcription of proneural basic helix-loop-helix factors, NEUROD1, NEUROG1 (significantly), and NEUROG2 (not significantly) was upregulated in medulloblastomas. The proportion of phosphorylated STAT3alpha relative to STAT3alpha was significantly greater in supratentorial PNETs than in medulloblastomas, indicating activation of the JAK/STAT3 pathway in supratentorial PNETs. These results indicate that supratentorial PNET predominantly has glial features and medulloblastoma largely follows a neuronal differentiation pattern. These divergent differentiation patterns may be related to the location and origin of each tumor.

Protein Tyrosine Phosphatases, TC-PTP, SHP1, and SHP2, Cooperate in Rapid Dephosphorylation of Stat3 in Keratinocytes Following UVB Irradiation

Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na3VO4 desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis.

Abstract 4007: Cooperative regulation of Stat3 signaling by TC-PTP, SHP1, and SHP2 in keratinocytes following UVB irradiation

Abstract Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na3VO4 desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45, the nuclear form of TC-PTP, SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are in part responsible for Stat3 dephosphorylation following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45, the isoform of TC-PTP expressed in keratinocytes, was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that simultaneous knockdown of all three phosphatases, TC45, SHP1, and SHP2, using RNAi recovered the level of phosphorylated Stat3 upon UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 also was initially decreased by UVB irradiation followed by a significant increase at later time points after UVB exposure. The expression patterns of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis. Supported by NIH grants CA105345, CA76520 and an Odyssey Fellowship to DJK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4007.

126. JAK/STAT SIGNALLING IN FOLLICULOGENESIS

Primordial follicle activation marks the first stage of pre-pubertal ovarian folliculogenesis, and is therefore fundamental to female fertility. Entry into development is initiated by a group of pleiotropic cytokines and growth factors, originating in and acting upon both the oocyte and granulosa support cells of the ovarian follicle through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling pathway. Pivotal to this process is the transcriptional regulation of target genes via STAT complexes and negative regulation by the Suppressors of Cytokine Signalling (SOCS) family of proteins. Preliminary evidence indicates that STAT3 facilitates the activation of primordial follicles, while SOCS4 counterbalances the activity of STAT3, mediating the controlled release of primordial follicles into the growing pool throughout reproductive life. Leukemia Inhibitory Factor (LIF) has been previously demonstrated as a key granulosa cell derived cytokine involved in inducing primordial follicle activation. Through both quantitative gene expression (qPCR) and immunoblotting we have demonstrated that LIF can significantly upregulate STAT3 mRNA production (~2-fold) as well as increase STAT3 protein phosphorylation within neonatal mouse ovarian explants culture. Furthermore, through the generation of a recombinant SOCS4 protein construct, and its use in subsequent protein-protein pull-downs, we were able to multiple targets involved in oocyte maturation, STAT3 interactions, and JAK/STAT signaling. These targets were also found to be significantly upregulated via qPCR analysis in neonatal mouse ovaries treated with LIF. These results support our current model for the involvement of STAT3 and SOCS4 in a basic negative feedback loop within the JAK/STAT signalling pathway that results in the regulation of primordial follicle activation and development.

Abstract B182: Single cell network profiling (SCNP) by flow cytometry as a tool to measure potency and selectivity of JAK/STAT inhibitors in PBMC and whole blood discrete cell subsets

Abstract Background: With the development of targeted therapeutics aimed at modulating signal transduction networks, there is a need for measuring the pharmacodynamic impact of these agents in relevant complex tissues at the single cell level. The ability to assess target coverage and selectivity is paramount - first, for selection of candidate molecules to progress through the drug development process and second, once a candidate drug moves into clinical testing, for guiding dose and schedule selection. Objectives: This study tested the ability to use single cell network profiling (SCNP), in which cells are perturbed with extracellular modulators and their response ascertained by multiparametric flow cytometry, to 1) measure the potency and selectivity of a panel of compounds with reported inhibitory effects on JAK/STAT signaling in whole blood versus fractionated peripheral blood mononuclear cells (PBMC) and 2) test the ability of SCNP to capture rare cells amid a heterogeneous population of cells. Methods: We measured modulation of JAK/STAT, MAPK and NFkB signaling pathways in cell subsets after in vitro exposure of whole blood and PBMC to GM-CSF (monocytes/neutrophils), IL-2 (T-cells), and CD40L (B-cells) in the presence and absence of known JAK inhibitors. For evaluation of SCNP in rare cells, phosphorylation of STAT proteins in response to IL-27 was examined on CD34+ CD11b− CD33− progenitors in healthy PBMCs. Results: The relative selectivity of JAK3 versus JAK2 inhibition was simultaneously measured by plotting p-Stat-5 inhibition curves from gated T cells (JAK3 mediated) and monocytes (JAK2 mediated). Notably, CP-690550, currently in clinical trials for a variety of autoimmune diseases, was 36-fold more potent at inhibiting JAK3 compared to JAK2 activity, which is consistent with published reports. In addition, off-target signaling effects outside the JAK/STAT pathway were observed (eg, Cucurbitacin I induced a dose-dependent increase in p-ERK in the monocyte subset and “Stat3 Inhibitor VII” blocked CD40L stimulated p-ERK and pNFkB p65 in B cells). Importantly, a significant decrease in potency in all compounds except CP-690550 was observed when studies were performed in whole blood, the clinically relevant tissue, versus fractionated PBMC. Notably, SCNP technology captured IL-27 induced p-Stat-1 and p-Stat-3 responses and inhibition by Pyridone 6 in CD34+ CD11b− CD33− progenitor cells, a very rare (&amp;lt;40 cells; 0.11% of the total live cells) cell population in PBMC. Conclusions: SCNP technology allowed for the evaluation of 1) compound activity in primary cells in a physiologically relevant environment (whole blood), 2) signal transduction target and off-target effects in relevant cell populations, and 3) robust IC50 determination from rare subpopulations (&amp;lt;100 cells). Therefore, this technology is well suited for application in oncology drug development where assessment of candidate drug effects on rare cell populations (eg, cancer stem cells, minimal residual disease, and circulating tumor cells) is crucial and permits the rational design of clinical trials based on biologically active dose rather than the traditional maximum tolerated dose (MTD) design, which is better suited for cytotoxic, non-targeted drugs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B182.

SOCS3 Deletion Promotes Optic Nerve Regeneration In Vivo

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.

The T Cell STAT Signaling Network Is Reprogrammed within Hours of Bacteremia via Secondary Signals

The delicate balance between protective immunity and inflammatory disease is challenged during sepsis, a pathologic state characterized by aspects of both a hyperactive immune response and immunosuppression. The events driven by systemic infection by bacterial pathogens on the T cell signaling network that likely control these responses have not been illustrated in great detail. We characterized how intracellular signaling within the immune compartment is reprogrammed at the single cell level when the host is challenged with a high level of pathogen. To accomplish this, we applied flow cytometry to measure the phosphorylation potential of key signal transduction proteins during acute bacterial challenge. We modeled the onset of sepsis by i.v. administration of avirulent strains of Listeria monocytogenes and Escherichia coli to mice. Within 6 h of bacterial challenge, T cells were globally restricted in their ability to respond to specific cytokine stimulations as determined by assessing the extent of STAT protein phosphorylation. Mechanisms by which this negative feedback response occurred included SOCS1 and SOCS3 gene up-regulation and IL-6-induced endocystosis of the IL-6 receptor. Additionally, macrophages were partially tolerized in their ability to respond to TLR agonists. Thus, in contrast to the view that there is a wholesale immune activation during sepsis, one immediate host response to blood-borne bacteria was induction of a refractory period during which leukocyte activation by specific stimulations was attenuated.

Investigation of constitutive and cytokine-induced expression of pSTAT 1, 3, and 5 in PBL of breast cancer patients

3042 Background: Although cytokines mediate their effects by latent transcription factors, STATs, their dysregulation has not been much investigated in peripheral blood lymphocytes (PBL) of breast cancer patients. As alterations in the JAK-STAT pathway contribute to immunosuppression in cancer patients in this study, aside from baseline-level, we investigated the level and cytokine-mediated activation of STAT1, 3, and 5 in healthy controls and breast cancer patients. Methods: PBL (5x106/ml) of breast cancer patients in all four clinical stages, as well as healthy controls, were treated in vitro with culture medium (CM) alone, IFNα (250 IU/ml CM) and IL-2 (500 IU/ml) for 24h at 37°C. Induction of phosphorylated STAT proteins was assessed in cellular lysates by Western blotting using monoclonal anti-STAT1, 3, and 5 antibodies. Treated PBL were also analyzed for IFNγ using an intracellular flow cytometry assay. Results: Our results indicate that untreated PBL of breast cancer patients, compared to healthy controls, express a significantly lower baseline level of pSTAT1, 3, and 5. The induction of pSTAT1 expression by IFNα, similar to the induction of pSTAT3 and pSTAT5 by IL-2, is significant for healthy controls and breast cancer patients, although the effect in patients is significantly below that for healthy controls. Moreover, the induction of pSTAT5 by IL-2 is of a higher magnitude than pSTAT3. PBL of breast cancer patients, compared to healthy controls, have a lower level of IFNγ. The production of IFNγ in PBL is enhanced by IFNα, as well as IL-2, although in breast cancer patients to a much lower level than in healthy controls. Conclusions: These results provide evidence that evaluation of STAT expression in PBL of breast cancer patients could be of diagnostic and therapeutic significance for cytokine-based therapy. No significant financial relationships to disclose.

Scorpion venom polypeptide accelerate irradiated hematopoietic cells proliferation

Objective To study the effects of Scorpion venom polypeptide (SVP) on the irradiated hematopoietic progenitor cells and the initial research of its mechanism. Methods and materials (1) MTT array was used to select the effective concentration of SVP that had proliferate action on the irradiated early hematopoietic cells (K562), just like the doses of experiment in vitro; (2) The male BALB/c mice were divided into NS control group, SVP IV group and SVP V group. After treatment and sublethal irradiation, the C-KIT and IL-6Rα levels of bone marrow cells were detected by immunohistochemistry and tissue array; (3) The bone marrow cells of the normal BALB/c mice, given to SVP IV and SVP V after different action times respectively, were taken to extract the total proteins inside the cell, the phosphorylated STAT3 protein levels in JAK-STAT signal transduction pathway were detected by Western blot array. Results (1) 30 mg/L SVP IV has an obvious effect to accelerate K562 cell proliferation; (2) The C-KIT and IL-6Rα expression on bone marrow cell surfaces in SVP IV and SVP V groups were negative (control with the saline group, p > 0.05); (3) The phosphorylated STAT3 protein levels in bone marrow cells of SVP IV group had a rise-and-fall trend within 30 min, while the test of SVP V group showed that the phosphorylated STAT3 protein levels obviously elevated after 30 min. Conclusions The results show that certain SVP IV concentration can protect the hematopoietic progenitor cells after irradiation, and the underlying mechanism of SVP accelerating the hematopoietic recovery in irradiated mice may be related to the activation of the JAK-STAT signal pathway.

SHD1 is a novel cytokine-inducible, negative feedback regulator of STAT5-dependent transcription

AbstractSTAT5 is a critical mediator of a variety of cytokine signaling whose transcriptional activity is regulated by associating with various proteins. During a search for STAT5-interacting proteins, we identified SHD1, a mammalian homologue of yeast gene Sac3, as a potential interacter. SHD1 was localized in the nucleus, and induced by cytokines that activate STAT5, such as erythropoietin, interleukin-2 (IL-2), or IL-3. SHD1 interacted specifically with STAT5A and STAT5B, and interestingly, it specifically repressed STAT5-dependent transcription in vitro without affecting the stability or phosphorylation of STAT5 protein. Gene disruption study revealed that T, B, or bone marrow cells from mice lacking SHD1 were hyperresponsive to T-cell–receptor engagement, or stimulation with various STAT5-activating cytokines. These results suggest that SHD1 is a novel cytokine-inducible negative feedback regulator of STAT5.

Inhibition of neurosphere proliferation by IFNγ but not IFNβ is coupled to neuronal differentiation

Interferons are produced following neural damage as part of the inflammatory response and may thus affect neural stem cell function. We compared the effects of interferon-γ and interferon-β on the proliferation and differentiation of adult murine neural progenitors. Both interferons inhibited neurosphere proliferation due to cell cycle arrest in G1 but only interferon-γ induced neuronal differentiation. Both interferons induced differential phosphorylation of STAT proteins and a modest and late upregulation of the cell cycle regulator p27 but not several other likely cell cycle regulators. Thus in neural progenitor cells, anti-proliferative effects of interferons are not necessarily linked to differentiation.

Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

Bioinformatic Analysis Reveals cRel as a Regulator of a Subset of Interferon-Stimulated Genes

Interferons (IFNs) are critical to the host innate immune response by inducing the expression of a family of early response genes, denoted as IFN-stimulated genes (ISGs). The role of tyrosine phosphorylation of STAT proteins in the transcription activation of ISGs is well-documented. Recent studies have indicated that other transcription factors (TFs) are likely to play a role in regulating ISG expression. Here, we describe a novel integrative approach that combines gene expression profiling, promoter sequence analysis, and literature mining to screen candidate regulatory factors in the IFN signal transduction pathway. Application of this method identified the nuclear factor kappaB (NFkappaB) protein, cRel, as a candidate regulatory factor for a subset of ISGs in mouse embryo fibroblasts. Chromatin immunoprecipitation (ChIP) and real-time PCR assays confirmed that cRel directly binds to the promoters of several ISGs, including Cxcl10, Isg15, Gbp2, Ifit3, and Ifi203, and regulates their expression. Thus, our studies identify cRel as an important TF for ISGs, and validate the approach of using Latent Semantic Indexing (LSI)-based methods to identify regulatory factors from microarray data.

Excessive phosphorylation of STATs leads to reduced chondrocyte proliferation and shortened bones in mucopolysaccharidosis VII mice

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease in which a deficiency in β‐glucuronidase activity results in glycosaminoglycan (GAG) accumulation in the growth plate, causing shortened long bones in various species through unclear mechanisms. Immunohistochemistry and RNA in‐situ hybridization showed that MPS VII mouse growth plates accumulate massive amounts of the GAG chondroitin‐4‐sulfate (C4S), have 53% of normal chondrocyte proliferation, express reduced levels of hypertrophic chondrocyte genes, and exhibit excessive phosphorylation of STAT proteins. In contrast, MPS I mice have less GAG storage and their bones and growth plates are nearly normal. Phosphorylated STATs are thought to mediate bone shortening in achondroplasia, a disease in which STATs are excessively phosphorylated by overactivation of fibroblast growth factor receptor 3 (FGFR3). Therefore, it was postulated that accumulation of C4S overstimulates FGFR3 in MPS VII. However, MPS VII mice that are FGFR3 deficient had shortened bones, suggesting that FGFR3 is not required for the bone defect. We propose that MPS VII bone disease is caused by a chondrocyte proliferation defect that may be mediated by STATs phosphorylated by an alternative pathway. Funding provided by Howard Hughes Medical Institute, Ryan Foundation, Mucopolysaccharidosis Society, and NIH (DK066448 and DK54481).