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A receptor-independent, cell-based JAK activation assay for screening for JAK3-specific inhibitors
Journal of Immunological Methods 354 (2010) 45–52
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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
A receptor-independent, cell-based JAK activation assay for screening for JAK3-specific inhibitors Keunhee Oh a,b,d, Kyeung Min Joo a, Yong Sik Jung d,e, Jaehwan Lee c,d, Heonjoong Kang c,d, Hee-Yoon Lee d,e, Dong-Sup Lee a,b,d,⁎ a b c d e
Laboratory of Immunology, Department of Anatomy, Seoul National University College of Medicine, Republic of Korea Transplantation Research Institute, Seoul National University College of Medicine, Republic of Korea Marine Biotechnology Laboratory, School of Earth and Environmental Sciences, Republic of Korea Center for Marine Natural Products Drug Discovery, Seoul National University, Seoul, Republic of Korea Department of Chemistry, KAIST, Daejeon, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 November 2009 Received in revised form 22 January 2010 Accepted 26 January 2010 Available online 4 February 2010 Keywords: JAK STAT TEL fusion protein JAK3 inhibitor
a b s t r a c t New immunosuppressive compounds with less systemic toxicity that could replace calcineurin inhibitors are urgently needed. For identification of specific inhibitors of JAK3, a potential new drug target, from large chemical libraries we developed a cell-based screening system. TEL–JAK fusion proteins composed of an oligomerization domain of TEL and kinase and/or pseudokinase domains of JAKs provided constitutive activation of JAKs without receiving a signal from the cytokine receptors. These fusion proteins also induced STAT5b phosphorylation in the absence of cytokine receptors. Both the kinase and pseudokinase domains of JAKs were required for full activation of the JAKs, and four copies of STAT5 response elements provided the greatest luciferase activity. The sensitivity and specificity of the system was evaluated using specific JAK3, JAK2, or MEK inhibitors. Thus, we generated a receptor-independent, cell-based selective screening system for specific JAK3 inhibitors, which is easily convertible to a high-throughput screening platform. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Transplantation is the final treatment option for patients with organ failure, and the long-term survival of patients depends heavily on the effective use of immunosuppressants (Meier-Kriesche et al., 2006). The use of cyclosporine A and
Abbreviations: JAK, Janus kinase; STAT, Signal transducer and activator of transcription; IL, Interleukin; γc chain, Common γ chain; TEL, Translocated Ets Leukemia/ETV6; GM-CSF, Granulocyte macrophage colony-stimulating factor; MEK, Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. ⁎ Corresponding author. Seoul National University, #28 Yongon-dong Chongno-gu, Seoul, 110-799, Republic of Korea. Tel.: + 82 10 2315 2227; fax: + 82 2 745 9528. E-mail address: [email protected] (D.-S. Lee). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.01.010
tacrolimus has tremendously increased the 5-year survival rate of organ-transplanted patients (Sam and Leehey, 2000). As calcineurin inhibitors prevent the induction of an immune tolerance mechanism by directly inhibiting the NFAT pathway (Heissmeyer et al., 2004), the inevitable continuous use of these drugs can lead to long-term immunosuppression and, in turn, recurrent infections. Additionally, these drugs can cause severe systemic side effects including nephrotoxicity, hypertension, and neurotoxicity (Liptak and Ivanyi, 2006). Therefore, new immunosuppressants with selectively targeting specific molecules with fewer side effects are required. Janus kinase 3 (JAK3) is an intracellular signaling component for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which share a common γ (γc) chain surface receptor. γc chain cytokines modulate the development, activation, proliferation, and survival of T, B, NK, and NKT cells (Rochman et al., 2009). As JAK3 expression is restricted to the immune system, JAK3 is a good molecular target for the development of novel
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immunosuppressants (O'Shea et al., 2004; Borie et al., 2004). Recently, JAK3-specific inhibitors such as CP-690,550 and PNU156804 have been shown to prolong graft survival in rodent models of heart transplantation and in cynomolgus monkeys receiving kidney transplants (Changelian et al., 2003; Borie et al., 2005; Stepkowski et al., 2002). Although JAK3-specific inhibitors might be good immunosuppressive reagents, there is no easy way to identify novel effective compounds in a large chemical library. In this study, we designed a cell-based, high-throughput screening system to identify JAK3-specific inhibitors. To develop a cytokine-independent system, we used a receptor-independent activation strategy naturally found in some leukemias (Lacronique et al., 2000, 1997). Fusion of the Etsfamily transcription factor TEL to JAK2 in an acute T cell lymphoblastic leukemia cell line has been shown to allow the cytokine-independent proliferation of hematopoietic cells (Lacronique et al., 1997). TEL–JAK fusion proteins bind each other through the TEL oligomerization domain, after which the closely localized JAKs are auto-phosphorylated and activated. The activated JAKs, in turn, phosphorylate STATs, and the phosphorylated STATs dimerize and translocate into the nucleus, where they function as transcription factors (Lacronique et al., 2000). Unlike most STATs that are recruited to cytokine receptors for activation (Leonard and O'Shea, 1998), STAT5a and STAT5b, can directly bind to JAK molecules in the absence of cytokine receptors (Fujitani et al., 1997) and be activated by them. By using constructs expressing the TEL– JAK fusion protein, STAT5b, and a reporter gene regulated by dimerized phospho-STAT5, our system provides 1) receptorindependent JAK activation; 2) receptor-independent, JAKdependent STAT activation; and 3) a sensitive activated-STAT reporter assay system for cell-based high-throughput screening for potential JAK3 inhibitors (Fig. 1).
2. Materials and methods 2.1. TEL–JAK fusion protein and STAT5b expression system Total RNA was isolated from human peripheral blood mononuclear cells using TRIZOL (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized using Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI) and random hexamers. One microliter of the first-strand cDNA reaction mixture was used in the subsequent PCR reactions. The primer sets used are: TEL, 5′-CTA GGC TAG CCC TGA TCT CTC TCG CTG TGA-3′/5′-AAT TGA ATT CAC AGT CTG CTA TTC TCC CAA TG-3′; JAK1 (JH1), 5′-GTA CGG TAC CCC AAC TGA AGT GGA CCC CAC-3′/5′-GGC CGC GGC CGC TGC TTC TTA TTT TAA AAG TGC TTC A -3′; JAK1 (JH2-1), 5′-GTA CGG TAC CCT CAA GAA GGA TCT GGT GCA-3′/5′-GGC CGC GGC CGC TGC TTC TTA TTT TAA AAG TGC TTC A-3′; JAK2 (JH1), 5′-AAT TGA ATT CGG TGC CCT AGG GTT TTC TGG T-3′/5′-GGC CGC GGC CGC TTT GGT CTC AGA ATG AAG GTC A-3′; JAK2 (JH2-1), 5′-AAT TGA ATT CGT GGA TGT ACC AAC CTC ACC A-3′/5′-GGC CGC GGC CGC TTT GGT CTC AGA ATG AAG GTC A-3′; JAK3 (JH1), 5′-AAT TGA ATT CAT TCG TGA CCT CAA TAG CCT CA-3′/ 5′-GGC CGC GGC CGC AAG GTC ACA CAG CCA GTC AA-3′; JAK3 (JH2-1), 5′-AAT TGA ATT CAA CCT GAT CGT GGT CCA GAG-3′/ 5′-GGC CGC GGC CGC AAG GTC ACA CAG CCA GTC AA-3′; STAT5b, 5′-GCC GAG CGA GAT TGT AAA CC-3′/5′-CCA CCA TGC ACA GAA ACA CT-3′. Amplified human TEL, JAK1(JH1), JAK1(JH2-1), JAK2(JH1), JAK2(JH2-1), JAK3(JH1), JAK3(JH21), and STAT5b DNA were cloned into the pGEM-T easy vector (Promega) and then subcloned into the pcDNA3.1(+) expression vector using the restriction enzyme sites, generating the TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2(JH2-1), TEL–JAK3(JH1), TEL–JAK3(JH2-1), and STAT5b expression plasmids. 2.2. Reporter system containing STAT5b responsive elements The STAT5 responsive element (RE) was synthesized as a fifteen-nucleotide, double-stranded DNA molecule (5′AATTTCCTGGAAATT-3′, BIONIX, Seoul, Korea) and blunt-end ligated into the SmaI-digested pTA-Luciferase vector (pTA-luc, Clontech, Mountain View, CA). The sequence and number (1, 2, or 4) of the STAT5 REs were verified by DNA sequencing using an ABI automated sequencer (Perkin Elmer, Boston, MA). 2.3. Transfection and JAK activity assay
Fig. 1. The receptor-independent, cell-based JAK3-specific inhibitor screening system. The JAK-STAT signaling pathway was constitutively activated through the use of a TEL–JAK fusion protein; receptor-independent, JAKdependent STAT activation; and a sensitive activated-STAT reporter assay.
The CV-1 (CCL-70) and TF-1 (CRL-2003) cell lines were used in this study. These cell lines were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Gaithersburg, MD) at 37 °C and 5% CO2. For the TF-1 cell line, 2 ng/ml recombinant human GM-CSF was added to the culture. Transfection was performed using the Lipofectamine 2000 transfection reagent (Invitrogen), following the manufacturer's recommended protocol. Briefly, 2 × 105 cells/well (in a 6-well plate) or 6 × 103 cells/well (in a 96-well plate) were transfected with various combinations of the TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL– JAK2(JH2-1), TEL–JAK3(JH1), TEL–JAK3(JH2-1), STAT5b, pTAluc, STAT5 RE(1)/pTA-luc, STAT5 RE(2)/pTA-luc, and STAT5
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RE(4)/pTA-luc plasmids. Twenty-four hours after transfection, protein tyrosine kinase inhibitors (CP-690,550, U0126, or AG490) were added at various concentrations, and the cells were incubated for an additional 24 h. Cell lysates were prepared and analyzed using a luciferase activity assay and western blot. Luciferase activity was assayed using a kit (Promega) and a Victor3 plate reader (Perkin Elmer) and normalized based on β-galactosidase activity. 2.4. Western blot Western blot was performed using standard protocols. Briefly, 48 h after transfection, cells were washed in cold PBS and lysed in RIPA buffer (0.5% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) containing a protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). After incubation in ice for 1 h, the supernatants were collected by centrifugation at 14,000 ×g for 5 min at 4 °C. The protein concentration was determined using BCA reagents (Pierce, Rockford, IL). A total of 10 μg of protein was resolved using 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) using electroblot. The membrane was probed with the specific primary and secondary antibodies. The polyclonal rabbit antiJAK1, -JAK2, -JAK3, -STAT5b, or -phospho-STAT5b (Tyr694) antibodies (Cell Signaling Technology, Danvers, MA) and mouse monoclonal anti-phosphotyrosine (Cell Signaling Technology) and anti-β-actin antibodies (Sigma) were used as primary antibodies. A horseradish peroxidase-conjugated goat anti-rabbit (Jackson Immunoresearch, West Grove, PA) or anti-mouse (Chemicon, Temecula, CA) IgG antibody were used as secondary antibodies. The signal was visualized using an enhanced chemiluminescence kit (Amersham, Piscataway, NJ). 2.5. Protein kinase inhibitors U0126 [1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] and AG490 (α-Cyano-(3,4-dihydroxy)N-benzylcinnamide) were purchased from Merck Bioscience (Darmstadt, Germany). 2.6. Synthesis of CP-690,550 CP-690,550 was synthesized through the coupling of (Nbenzyl-4-methyl-piperidin-3-yl)methylamine and 4-chloropyrrolo[2,3-d]pyrimidine in the presence of potassium carbonate, followed by hydrogenolytic debenzylation and subsequent cyanoacetamide formation using cyanoacetic acid 2,5-dioxopyrrolidine-1-yl ester. (N-benzyl-4-methyl-piperidin-3-yl)methylamine was prepared from 4-picolin following the literature preparation. 4-Picolin was quarternized with benzyl chloride to produce a pyridinium salt that was reduced with sodium borohydride. The tetrahydropicolin compound was then subjected to a sequence of hydroboration–oxidation, Swern's oxidation, and reductive amination with methylamine. Resolution of the amine with a tartaric acid derivative resulted in enatiomerically pure (N-benzyl-4methyl-piperidin-3-yl)methylamine.
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3. Results 3.1. TEL–JAK fusion protein construction Cytokine-independent JAK activation was induced by constructing TEL–JAK fusion proteins. The oligomerization domain of TEL (amino acids 1–336 of human TEL, Fig. 2A) and the protein tyrosine kinase domain (JH1) and/or pseudokinase domain (JH2) of different JAKs (Fig. 2A) were fused, producing two different constructs for each JAK [TEL–JAK1 (JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2(JH2-1), TEL–JAK3(JH1), and TEL–JAK3(JH2-1)]. TEL–JAK expression constructs were established by subcloning the TEL–JAK (JH1 or JH2-1) fusions into the pcDNA3.1(+) expression vector (Fig. 2B). Fusion protein expression and JAK phosphorylation were confirmed by transfecting the fusion constructs into CV1 cells (monkey kidney fibroblasts) and performing a western blot on extracts from the transfected cells with an anti-JAK antibody and an anti-phosphotyrosine antibody. The 78.6kDa TEL–JAK3(JH1) fusion protein and the 110.6-kDa TEL– JAK3(JH2-1) fusion protein, both with phosphorylated tyrosine residues, were identified (Fig. 2C). The expression and phosphorylation of TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL– JAK2(JH1), and TEL–JAK2(JH2-1) was similarly confirmed using western blot (data not shown). 3.2. STAT5 responsive element reporter system The human STAT5b gene was cloned and inserted into an expression vector (Fig. 3A). The STAT reporter system was generated by cloning 1, 2, or 4 copies of the STAT5 REs (Soldaini et al., 2000), each separated by six nucleotides, into the promoter region of the pTA luciferase vector (RE(1), RE (2), and RE(4), respectively) (Fig. 3B). To evaluate the optimal number of REs in the reporter system, STAT5b and STAT5 RE/pTA-luc were transfected into TF-1, a cytokinedependent erythroblastic cell line. Transfected TF-1 cells were cultured in the presence of 2 ng/ml of GM-CSF for 48 h, and luciferase activity was monitored. Compared with the control, which was transfected only with STAT5b, five times greater luciferase activity was detected in the cells cotransfected with the STAT5b expression plasmid and STAT5 RE(4)/pTA-luc (Fig. 3C). In contrast, cells transfected with the STAT5b expression plasmid plus STAT5 RE(1)/pTA-luc or STAT5 RE(2)/pTA-luc showed a similar level of luciferase activity compared with the control (Fig. 3C). These results indicate that four STAT5 REs allow for the most efficient luciferase expression in response to the binding of dimerized STAT5b. 3.3. Cell-based, JAK3-specific inhibitor screening system To generate a receptor-independent, JAK-dependent STAT activation system, CV-1 cells were co-transfected with 1) TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2 (JH2-1), TEL–JAK3(JH1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(1), RE(2), or RE(4)/pTA-luc. The transfected cells were cultured for 48 h, and luciferase activity was measured. The expression of the fusion proteins including both the JH2 and JH1 domains of JAK3 showed higher luciferase activity than proteins containing the JH1 domain alone (Fig. 3D).
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Fig. 2. Expression and activation of the TEL–JAK fusion proteins. (A) Human TEL, JAK1, JAK2, and JAK3 gene structure. (B) The TEL–JAK fusion constructs. (C) The expression of the TEL–JAK3 fusion protein (left panel) and the activation of JAK3 in the TEL–JAK3 fusion protein (right panel) in the CV-1 cell line were confirmed using western blot with an anti-JAK3 antibody and an anti-phosphotyrosine antibody, respectively. TEL–JAK3(JH1) fusion protein, 78.6 kDa; TEL–JAK3(JH2-1) fusion protein, 110.6 kDa.
Consistent with the results in TF-1 cells, STAT5 RE(4) showed the greatest signal compared with either STAT5 RE(1) or STAT5 RE(2). Additionally, the luciferase activity in CV-1 cells expressing the TEL–JAK3 fusion protein was much higher than that in TF-1 cells, which require the cytokine-dependent activation of endogenous JAKs. The greatest luciferase signal was observed in CV-1 cells transfected with TEL–JAK3(JH2-1) and STAT5 RE(4), and therefore, we used TEL–JAK(JH2-1) and STAT5 RE(4) in the development of the cell-based, highthroughput system to screen for JAK3-specific inhibitors. Importantly, the endogenous JAKs and STATs did not appear to induce luciferase expression in our system (Fig. 3D).
3.4. Sensitivity and specificity of the cell-based, JAK3-specific inhibitor screening system CP-690,550, a JAK3-specific inhibitor (Changelian et al., 2003), was used to evaluate the sensitivity of the system. CV1 cells were co-transfected with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc and cultured for 48 h. Various concentrations of CP-690,550 (101–107 nM) were added for the final 24 h. The IC50 of CP-690,550 for JAK3 was calculated as 12.4 μM (Fig. 4A). The inhibitory activity of CP-690,550 was not due to cytotoxic effects, as the LD50 of CP-690,550 was 1.93 mM (Fig. 4B). The inhibition of JAK3 and downstream STAT5b phosphorylation by CP-690,550 was also confirmed using western blot. With increasing concentrations of CP690,550, the amount of phosphorylated STAT5b decreased, and at concentrations of CP-690,550 over 10 μM, this signal disappeared (Fig. 4C). CP-690,550 did not affect the expression of the transfected STAT5b and TEL–JAK fusion proteins (Fig. 4C). The CV-1 cells used in the assay expressed little
endogenous JAK3 and only a small amount of STAT5b (Fig. 4C). Phosphorylated STAT5b was hardly detected using western blot in the TEL–JAK3(JH2-1) construct only-group or the STAT5 RE(4)/pTA-luc construct only-transfected group (Fig. 4C), indicating that the endogenous STAT5b in CV-1 cells had little effect on our system. The TEL–JAK3(JH2-1)-STAT5bSTAT5 RE(4)/pTA-luc system and its inhibition by CP-690,550 was confirmed using a luciferase assay (Fig. 4D). The specificity of the system was evaluated using the JAK1 and JAK2 constructs and CP-690,550 (100 μM) in the assay. CV-1 cells were co-transfected with 1) TEL–JAK1(JH2-1), TEL–JAK2(JH2-1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(4)/pTA-luc. The inhibitory activity of CP-690,550 was only observed in the cells transfected with TEL–JAK3 (JH2-1); no inhibition was seen with the JAK2 or JAK1 fusion constructs (Fig. 5A). The specificity of the JAK3 system was further evaluated using U0126, an MEK-1 and MEK-2 protein tyrosine kinase inhibitor (Duncia et al., 1998) and AG490, a JAK2-specific inhibitor (Miyamoto et al., 2001) in the assay. JAK3-specific luciferase activity was not inhibited by U0126 (Fig. 5B) or AG490 (Fig. 5C). Furthermore, 105 nM AG490 showed overt cytotoxicity to CV-1 cells (data not shown).
4. Discussion Even with their severe systemic toxicity, calcineurin inhibitors have been the most widely used compounds to suppress unwanted immune responses, such as in the inhibition of graft rejection and in the treatment of autoimmune disease (Meier-Kriesche et al., 2006; Sam and Leehey, 2000). The introduction of novel immunosuppressants that could replace or reduce the dosage of calcineurin inhibitors
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Fig. 3. Construction of a cell-based, JAK3-specific inhibitor screening system. (A) Structure of the human STAT5b gene. (B) The STAT5 RE/pTA-luc DNA constructs. STAT5 RE(1), RE(2), and RE(4) contained 1, 2, and 4 copies of the STAT5 RE sequence, respectively. (C) The efficacy of the STAT5 RE/pTA-luc constructs with 1, 2, or 4 REs was evaluated by co-transfecting cytokine-dependent TF-1 cells with a STAT5b expression plasmid and either STAT5 RE(1), RE(2), or RE(4). The transfected TF-1 cells were cultured in the presence of 2 ng/ml of GM-CSF, and a luciferase activity assay was performed. (D) The cell-based, JAK3-specific inhibitor screening system was made by co-transfecting CV-1 cells with 1) TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2(JH2-1), TEL–JAK3(JH1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(1), RE(2), or RE(4)/pTA-luc in a 96-well plate. Each combination was analyzed in triplicate, and the data shown are representative of three independent experiments.
could alleviate the lethal systemic side effects of these drugs and provide a better outcome for the patients. γc chain cytokines have been shown to play a critical role during lymphocyte development, activation, survival, and homeostasis (Schmitt and Zuniga-Pflucker, 2005; Gattinoni et al., 2005; Kaech et al., 2003) and have been implicated in critical aspects of immune regulation such as T cell anergy, memory T cell survival and homeostasis, and regulatory T cell development and function (Grundstrom et al., 2000; Tan et al., 2002; Wan and Flavell, 2006). While the clinical proof of concept for the use of JAK3 inhibitors as a putative immunosuppressant has been promising (Kremer et al., 2009), evaluating whether JAK3 inhibitors also affect the induction of tolerance would be challenging.
A cell-based system is essential in finding specific signaling inhibitors that work in the context of molecular networks that normally exist inside a cell. During cytokine signaling, receptor binding is generally required for both JAK phosphorylation (Chen et al., 1997; Zhao et al., 1995) and STAT phosphorylation (Naeger et al., 1999; Yang et al., 1996). Following expression in CV-1 cells, a cytokine-independent cell line, the TEL fusion protein in our study efficiently promoted receptor-independent JAK phosphorylation for JAK1 and JAK3, as well as JAK2, which has been shown to be fused to TEL in some leukemias (Lacronique et al., 1997). Also our strategy of using direct interactions with JAK and STAT through the kinase and/or pseudokinase domain of the JAKs efficiently induced receptor-independent STAT
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Fig. 4. Sensitivity of the cell-based, JAK3-specific inhibitor screening system. (A) CP-690,550, a JAK3-specific inhibitor, was used to test the sensitivity of the system. CV-1 cells were co-transfected with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc and cultured with various concentrations of CP-690,550 for 48 h. The luciferase activity in the transfected cells was measured and compared with controls that were transfected, but treated with PBS. Each sample was tested in triplicate, and the data shown are representative of three independent experiments. (B) The cytotoxicity of CP-690,550 was tested by co-transfecting CV-1 cells with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc. The transfected cells were cultured in various concentrations of CP-690,550 for 48 h, and the viability was checked using a MTT assay. Each sample was tested in triplicate. (C) The system was validated using western blot. Different combinations of TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc were co-transfected into CV-1 cells, and the cells were cultured in the indicated concentration of CP-690,550 for 48 h. The expression of STAT5b and the TEL–JAK3 fusion protein and the activation of STAT5b were analyzed using anti-STAT5b, anti-JAK3, and anti-phospho-STAT5b antibodies, respectively. (D) Different combinations of TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc were co-transfected into CV-1 cells The transfected cells were cultured with 100 μM CP-690,550 or PBS for 48 h, after which a luciferase activity assay was performed. Each sample was tested in triplicate, and the data shown are representative of three independent experiments.
activation. The relative role of the kinase (JH1) and pseudokinase (JH2) domains in the direct interaction with STAT5s is controversial. In our assay, while the luciferase activity was similar between the cells expressing JAK1(JH1) and JAK1(JH2-1), the activity was 100% and 30% greater in JAK2(JH2-1) compared to JAK2(JH1) and JAK3(JH2-1) compared to JAK3(JH1), respectively. In contrast to a previous report demonstrating the exclusive role of the pseudokinase (JH2) domain in direct STAT5 binding in a cell-free protein binding assay, our result suggests that, in this cell-based system, the JH1 domain played a more dominant role in STAT5 activation, especially for JAK1 and JAK3 (Fujitani et al., 1997). By co-transfecting artificially constructed JAK signaling components into a cytokine-independent cell line, we generated a receptor-independent, cell-based assay system to measure JAK activity (Fig. 1). The IC50 of CP-690,550 for JAK3 (12.4 μM) was comparable with the IC50 of CP-690,550 in the inhibition of anti-CD3 and anti-CD28-mediated activation of Jurkat cells (7.8 μM; measured using IL-2 production) (Changelian et al., 2003). This demonstrates
that our cell-based co-transfection assay system showed similar sensitivity with other cell-based functional assays. The IC50 of CP-690,550 for the JAK1 and JAK2 constructs were 775.1 μM and 214.4 μM, respectively (our unpublished data), and this paralleled, with some differences, the IC50 of CP690,550 measured in a cell-free kinase activity assay system (Changelian et al., 2003). It will be very important to use a cell-based assay system to see the physiological effects of the inhibitors. The CV-1 cells used in our assay system showed minimal endogenous STAT5b and JAK3 expression (Fig. 4C) and little background activity of endogenous JAKs as the transfected STAT5b was not phosphorylated in the absence of the co-transfected TEL fusion protein (Fig. 4C). This very low background activity of the endogenous JAK3 or STAT5b was confirmed using a luciferase assay (Fig. 4D). Therefore, these cells would be ideal for our receptor-independent, cell-based JAK assay system. Our cell-based JAK3-specific inhibitor screening system has several advantages. The cytotoxicity of the drugs was monitored simultaneously, and other factors, such as the bioavailability of drugs inside different subcellular
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compartments and the effect of intracellular environments, were reflected in the assay. Therefore, our assay provides a physiological measurement of the JAK3-inhibiting activity of the compound in the context of the normal cellular milieu. As our system can directly test the JAK1-, JAK2-, or JAK3inhibiting capacity of the chemicals simultaneously, specific JAK1 or JAK2 inhibitors can easily be identified. Importantly, JAK2 inhibitors have recently been focused on as novel antileukemia drugs (Atallah and Verstovsek, 2009) and JAK1/ JAK2 inhibitor screening based on cell lines and primary cells from polycythemia vera patients harboring activating JAK2 mutations was recently reported (Pardanani et al., 2009). In addition, our screening method is simple and requires no exogenous cytokines or endogenous receptors, which makes it easily automatable in 96-well or 384-well formats. A virtual screening method to identify novel JAK3 inhibitors was recently reported and this approach may be very useful for pre-screening compounds from the large chemical libraries before performing our cell-based assays (Chen et al., 2008). Acknowledgements This research was supported by grants from MarineBio Technology Project funded by Ministry of Land, Transport and Maritime Affairs and from Korea Science and Engineering Foundation funded by the Ministry of Science and Technology (No. M1064152000106N415200110) and from Korea Healthcare technology R&D Project funded by Ministry for Health, Welfare & Family Affairs (No. A084022). References
Fig. 5. Specificity of the cell-based, JAK3-specific inhibitor screening system. (A) CV-1 cells were co-transfected with 1) TEL–JAK1(JH2-1), TEL–JAK2(JH2-1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(4)/ pTA-luc and cultured with 100 μM CP-690,550 or PBS for 48 h. A luciferase activity assay was then performed. Each sample was tested in triplicate, and the data shown are representative of three independent experiments. (B–C) CV-1 cells were co-transfected with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc and cultured with various concentrations of (B) U0126 (a MEK inhibitor) or (C) AG490 (a JAK2 specific inhibitor) for 48 h. Luciferase activity assay was then performed. The luciferase activities were compared with the transfected, PBS-treated controls. Each sample was analyzed in triplicate.
Atallah, E., Verstovsek, S., 2009. Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms. Expert Rev. Anticancer Ther. 9, 663. Borie, D.C., O'Shea, J.J., Changelian, P.S., 2004. JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants. Trends Mol. Med. 10, 532. Borie, D.C., Changelian, P.S., Larson, M.J., Si, M.S., Paniagua, R., Higgins, J.P., Holm, B., Campbell, A., Lau, M., Zhang, S., Flores, M.G., Rousvoal, G., Hawkins, J., Ball, D.A., Kudlacz, E.M., Brissette, W.H., Elliott, E.A., Reitz, B.A., Morris, R.E., 2005. Immunosuppression by the JAK3 inhibitor CP-690550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates. Transplantation 79, 791. Changelian, P.S., Flanagan, M.E., Ball, D.J., Kent, C.R., Magnuson, K.S., Martin, W.H., Rizzuti, B.J., Sawyer, P.S., Perry, B.D., Brissette, W.H., McCurdy, S.P., Kudlacz, E.M., Conklyn, M.J., Elliott, E.A., Koslov, E.R., Fisher, M.B., Strelevitz, T.J., Yoon, K., Whipple, D.A., Sun, J., Munchhof, M.J., Doty, J.L., Casavant, J.M., Blumenkopf, T.A., Hines, M., Brown, M.F., Lillie, B.M., Subramanyam, C., Shang-Poa, C., Milici, A.J., Beckius, G.E., Moyer, J.D., Su, C., Woodworth, T.G., Gaweco, A.S., Beals, C.R., Littman, B.H., Fisher, D.A., Smith, J.F., Zagouras, P., Magna, H.A., Saltarelli, M.J., Johnson, K.S., Nelms, L.F., Des Etages, S.G., Hayes, L.S., Kawabata, T.T., Finco-Kent, D., Baker, D.L., Larson, M., Si, M.S., Paniagua, R., Higgins, J., Holm, B., Reitz, B., Zhou, Y.J., Morris, R.E., O'Shea, J.J., Borie, D.C., 2003. Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor. Science 302, 875. Chen, M., Cheng, A., Chen, Y.Q., Hymel, A., Hanson, E.P., Kimmel, L., Minami, Y., Taniguchi, T., Changelian, P.S., O'Shea, J.J., 1997. The amino terminus of JAK3 is necessary and sufficient for binding to the common gamma chain and confers the ability to transmit interleukin 2-mediated signals. Proc. Natl. Acad. Sci. U S A. 94, 6910. Chen, X., Wilson, L.J., Malaviya, R., Argentieri, R.L., Yang, S.M., 2008. Virtual screening to successfully identify novel Janus kinase 3 inhibitors: a sequential focused screening approach. J. Med. Chem. 51, 7015. Duncia, J.V., Santella 3rd., J.B., Higley, C.A., Pitts, W.J., Wityak, J., Frietze, W.E., Rankin, F.W., Sun, J.H., Earl, R.A., Tabaka, A.C., Teleha, C.A., Blom, K.F., Favata, M.F., Manos, E.J., Daulerio, A.J., Stradley, D.A., Horiuchi, K., Copeland, R.A., Scherle, P.A., Trzaskos, J.M., Magolda, R.L., Trainor, G.L., Wexler, R.R., Hobbs, F.W., Olson, R.E., 1998. MEK inhibitors: the
52
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chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorg. Med. Chem. Lett. 8, 2839. Fujitani, Y., Hibi, M., Fukada, T., Takahashi-Tezuka, M., Yoshida, H., Yamaguchi, T., Sugiyama, K., Yamanaka, Y., Nakajima, K., Hirano, T., 1997. An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT. Oncogene 14, 751. Gattinoni, L., Finkelstein, S.E., Klebanoff, C.A., Antony, P.A., Palmer, D.C., Spiess, P.J., Hwang, L.N., Yu, Z., Wrzesinski, C., Heimann, D.M., Surh, C.D., Rosenberg, S.A., Restifo, N.P., 2005. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J. Exp. Med. 202, 907. Grundstrom, S., Dohlsten, M., Sundstedt, A., 2000. IL-2 unresponsiveness in anergic CD4+ T cells is due to defective signaling through the common gamma-chain of the IL-2 receptor. J. Immunol. 164, 1175. Heissmeyer, V., Macian, F., Im, S.H., Varma, R., Feske, S., Venuprasad, K., Gu, H., Liu, Y.C., Dustin, M.L., Rao, A., 2004. Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat. Immunol. 5, 255. Kaech, S.M., Tan, J.T., Wherry, E.J., Konieczny, B.T., Surh, C.D., Ahmed, R., 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4, 1191. Kremer, J.M., Bloom, B.J., Breedveld, F.C., Coombs, J.H., Fletcher, M.P., Gruben, D., Krishnaswami, S., Burgos-Vargas, R., Wilkinson, B., Zerbini, C.A., Zwillich, S.H., 2009. The safety and efficacy of a JAK inhibitor in patients with active rheumatoid arthritis: results of a double-blind, placebocontrolled phase IIA trial of three dosage levels of CP-690550 versus placebo. Arthritis Rheum. 60, 1895. Lacronique, V., Boureux, A., Valle, V.D., Poirel, H., Quang, C.T., Mauchauffe, M., Berthou, C., Lessard, M., Berger, R., Ghysdael, J., Bernard, O.A., 1997. A TEL–JAK2 fusion protein with constitutive kinase activity in human leukemia. Science 278, 1309. Lacronique, V., Boureux, A., Monni, R., Dumon, S., Mauchauffe, M., Mayeux, P., Gouilleux, F., Berger, R., Gisselbrecht, S., Ghysdael, J., Bernard, O.A., 2000. Transforming properties of chimeric TEL–JAK proteins in Ba/F3 cells. Blood 95, 2076. Leonard, W.J., O'Shea, J.J., 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16, 293. Liptak, P., Ivanyi, B., 2006. Primer: histopathology of calcineurin-inhibitor toxicity in renal allografts. Nat. Clin. Pract. Nephrol. 2, 398. Meier-Kriesche, H.U., Li, S., Gruessner, R.W., Fung, J.J., Bustami, R.T., Barr, M.L., Leichtman, A.B., 2006. Immunosuppression: evolution in practice and trends, 1994–2004. Am. J. Transplant. 6, 1111. Miyamoto, N., Sugita, K., Goi, K., Inukai, T., Lijima, K., Tezuka, T., Kojika, S., Nakamura, M., Kagami, K., Nakazawa, S., 2001. The JAK2 inhibitor AG490
predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome. Leukemia 15, 1758. Naeger, L.K., McKinney, J., Salvekar, A., Hoey, T., 1999. Identification of a STAT4 binding site in the interleukin-12 receptor required for signaling. J. Biol. Chem. 274, 1875. O'Shea, J.J., Pesu, M., Borie, D.C., Changelian, P.S., 2004. A new modality for immunosuppression: targeting the JAK/STAT pathway. Nat. Rev. Drug Discov. 3, 555. Pardanani, A., Lasho, T., Smith, G., Burns, C.J., Fantino, E., Tefferi, A., 2009. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients. Leukemia 23, 1441. Rochman, Y., Spolski, R., Leonard, W.J., 2009. New insights into the regulation of T cells by gamma(c) family cytokines. Nat. Rev. Immunol. 9, 480. Sam, R., Leehey, D.J., 2000. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N. Engl. J. Med. 342, 1837. Schmitt, T.M., Zuniga-Pflucker, J.C., 2005. Thymus-derived signals regulate early T-cell development. Crit. Rev. Immunol. 25, 141. Soldaini, E., John, S., Moro, S., Bollenbacher, J., Schindler, U., Leonard, W.J., 2000. DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites. Mol. Cell. Biol. 20, 389. Stepkowski, S.M., Erwin-Cohen, R.A., Behbod, F., Wang, M.E., Qu, X., Tejpal, N., Nagy, Z.S., Kahan, B.D., Kirken, R.A., 2002. Selective inhibitor of Janus tyrosine kinase 3, PNU156804, prolongs allograft survival and acts synergistically with cyclosporine but additively with rapamycin. Blood 99, 680. Tan, J.T., Ernst, B., Kieper, W.C., LeRoy, E., Sprent, J., Surh, C.D., 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med. 195, 1523. Wan, Y.Y., Flavell, R.A., 2006. The roles for cytokines in the generation and maintenance of regulatory T cells. Immunol. Rev. 212, 114. Yang, C.H., Shi, W., Basu, L., Murti, A., Constantinescu, S.N., Blatt, L., Croze, E., Mullersman, J.E., Pfeffer, L.M., 1996. Direct association of STAT3 with the IFNAR-1 chain of the human type I interferon receptor. J. Biol. Chem. 271, 8057. Zhao, Y., Wagner, F., Frank, S.J., Kraft, A.S., 1995. The amino-terminal portion of the JAK2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor beta c chain. J. Biol. Chem. 270, 13814.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
A receptor-independent, cell-based JAK activation assay for screening for JAK3-specific inhibitors Keunhee Oh a,b,d, Kyeung Min Joo a, Yong Sik Jung d,e, Jaehwan Lee c,d, Heonjoong Kang c,d, Hee-Yoon Lee d,e, Dong-Sup Lee a,b,d,⁎ a b c d e
Laboratory of Immunology, Department of Anatomy, Seoul National University College of Medicine, Republic of Korea Transplantation Research Institute, Seoul National University College of Medicine, Republic of Korea Marine Biotechnology Laboratory, School of Earth and Environmental Sciences, Republic of Korea Center for Marine Natural Products Drug Discovery, Seoul National University, Seoul, Republic of Korea Department of Chemistry, KAIST, Daejeon, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 November 2009 Received in revised form 22 January 2010 Accepted 26 January 2010 Available online 4 February 2010 Keywords: JAK STAT TEL fusion protein JAK3 inhibitor
a b s t r a c t New immunosuppressive compounds with less systemic toxicity that could replace calcineurin inhibitors are urgently needed. For identification of specific inhibitors of JAK3, a potential new drug target, from large chemical libraries we developed a cell-based screening system. TEL–JAK fusion proteins composed of an oligomerization domain of TEL and kinase and/or pseudokinase domains of JAKs provided constitutive activation of JAKs without receiving a signal from the cytokine receptors. These fusion proteins also induced STAT5b phosphorylation in the absence of cytokine receptors. Both the kinase and pseudokinase domains of JAKs were required for full activation of the JAKs, and four copies of STAT5 response elements provided the greatest luciferase activity. The sensitivity and specificity of the system was evaluated using specific JAK3, JAK2, or MEK inhibitors. Thus, we generated a receptor-independent, cell-based selective screening system for specific JAK3 inhibitors, which is easily convertible to a high-throughput screening platform. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Transplantation is the final treatment option for patients with organ failure, and the long-term survival of patients depends heavily on the effective use of immunosuppressants (Meier-Kriesche et al., 2006). The use of cyclosporine A and
Abbreviations: JAK, Janus kinase; STAT, Signal transducer and activator of transcription; IL, Interleukin; γc chain, Common γ chain; TEL, Translocated Ets Leukemia/ETV6; GM-CSF, Granulocyte macrophage colony-stimulating factor; MEK, Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. ⁎ Corresponding author. Seoul National University, #28 Yongon-dong Chongno-gu, Seoul, 110-799, Republic of Korea. Tel.: + 82 10 2315 2227; fax: + 82 2 745 9528. E-mail address: [email protected] (D.-S. Lee). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.01.010
tacrolimus has tremendously increased the 5-year survival rate of organ-transplanted patients (Sam and Leehey, 2000). As calcineurin inhibitors prevent the induction of an immune tolerance mechanism by directly inhibiting the NFAT pathway (Heissmeyer et al., 2004), the inevitable continuous use of these drugs can lead to long-term immunosuppression and, in turn, recurrent infections. Additionally, these drugs can cause severe systemic side effects including nephrotoxicity, hypertension, and neurotoxicity (Liptak and Ivanyi, 2006). Therefore, new immunosuppressants with selectively targeting specific molecules with fewer side effects are required. Janus kinase 3 (JAK3) is an intracellular signaling component for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which share a common γ (γc) chain surface receptor. γc chain cytokines modulate the development, activation, proliferation, and survival of T, B, NK, and NKT cells (Rochman et al., 2009). As JAK3 expression is restricted to the immune system, JAK3 is a good molecular target for the development of novel
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immunosuppressants (O'Shea et al., 2004; Borie et al., 2004). Recently, JAK3-specific inhibitors such as CP-690,550 and PNU156804 have been shown to prolong graft survival in rodent models of heart transplantation and in cynomolgus monkeys receiving kidney transplants (Changelian et al., 2003; Borie et al., 2005; Stepkowski et al., 2002). Although JAK3-specific inhibitors might be good immunosuppressive reagents, there is no easy way to identify novel effective compounds in a large chemical library. In this study, we designed a cell-based, high-throughput screening system to identify JAK3-specific inhibitors. To develop a cytokine-independent system, we used a receptor-independent activation strategy naturally found in some leukemias (Lacronique et al., 2000, 1997). Fusion of the Etsfamily transcription factor TEL to JAK2 in an acute T cell lymphoblastic leukemia cell line has been shown to allow the cytokine-independent proliferation of hematopoietic cells (Lacronique et al., 1997). TEL–JAK fusion proteins bind each other through the TEL oligomerization domain, after which the closely localized JAKs are auto-phosphorylated and activated. The activated JAKs, in turn, phosphorylate STATs, and the phosphorylated STATs dimerize and translocate into the nucleus, where they function as transcription factors (Lacronique et al., 2000). Unlike most STATs that are recruited to cytokine receptors for activation (Leonard and O'Shea, 1998), STAT5a and STAT5b, can directly bind to JAK molecules in the absence of cytokine receptors (Fujitani et al., 1997) and be activated by them. By using constructs expressing the TEL– JAK fusion protein, STAT5b, and a reporter gene regulated by dimerized phospho-STAT5, our system provides 1) receptorindependent JAK activation; 2) receptor-independent, JAKdependent STAT activation; and 3) a sensitive activated-STAT reporter assay system for cell-based high-throughput screening for potential JAK3 inhibitors (Fig. 1).
2. Materials and methods 2.1. TEL–JAK fusion protein and STAT5b expression system Total RNA was isolated from human peripheral blood mononuclear cells using TRIZOL (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized using Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI) and random hexamers. One microliter of the first-strand cDNA reaction mixture was used in the subsequent PCR reactions. The primer sets used are: TEL, 5′-CTA GGC TAG CCC TGA TCT CTC TCG CTG TGA-3′/5′-AAT TGA ATT CAC AGT CTG CTA TTC TCC CAA TG-3′; JAK1 (JH1), 5′-GTA CGG TAC CCC AAC TGA AGT GGA CCC CAC-3′/5′-GGC CGC GGC CGC TGC TTC TTA TTT TAA AAG TGC TTC A -3′; JAK1 (JH2-1), 5′-GTA CGG TAC CCT CAA GAA GGA TCT GGT GCA-3′/5′-GGC CGC GGC CGC TGC TTC TTA TTT TAA AAG TGC TTC A-3′; JAK2 (JH1), 5′-AAT TGA ATT CGG TGC CCT AGG GTT TTC TGG T-3′/5′-GGC CGC GGC CGC TTT GGT CTC AGA ATG AAG GTC A-3′; JAK2 (JH2-1), 5′-AAT TGA ATT CGT GGA TGT ACC AAC CTC ACC A-3′/5′-GGC CGC GGC CGC TTT GGT CTC AGA ATG AAG GTC A-3′; JAK3 (JH1), 5′-AAT TGA ATT CAT TCG TGA CCT CAA TAG CCT CA-3′/ 5′-GGC CGC GGC CGC AAG GTC ACA CAG CCA GTC AA-3′; JAK3 (JH2-1), 5′-AAT TGA ATT CAA CCT GAT CGT GGT CCA GAG-3′/ 5′-GGC CGC GGC CGC AAG GTC ACA CAG CCA GTC AA-3′; STAT5b, 5′-GCC GAG CGA GAT TGT AAA CC-3′/5′-CCA CCA TGC ACA GAA ACA CT-3′. Amplified human TEL, JAK1(JH1), JAK1(JH2-1), JAK2(JH1), JAK2(JH2-1), JAK3(JH1), JAK3(JH21), and STAT5b DNA were cloned into the pGEM-T easy vector (Promega) and then subcloned into the pcDNA3.1(+) expression vector using the restriction enzyme sites, generating the TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2(JH2-1), TEL–JAK3(JH1), TEL–JAK3(JH2-1), and STAT5b expression plasmids. 2.2. Reporter system containing STAT5b responsive elements The STAT5 responsive element (RE) was synthesized as a fifteen-nucleotide, double-stranded DNA molecule (5′AATTTCCTGGAAATT-3′, BIONIX, Seoul, Korea) and blunt-end ligated into the SmaI-digested pTA-Luciferase vector (pTA-luc, Clontech, Mountain View, CA). The sequence and number (1, 2, or 4) of the STAT5 REs were verified by DNA sequencing using an ABI automated sequencer (Perkin Elmer, Boston, MA). 2.3. Transfection and JAK activity assay
Fig. 1. The receptor-independent, cell-based JAK3-specific inhibitor screening system. The JAK-STAT signaling pathway was constitutively activated through the use of a TEL–JAK fusion protein; receptor-independent, JAKdependent STAT activation; and a sensitive activated-STAT reporter assay.
The CV-1 (CCL-70) and TF-1 (CRL-2003) cell lines were used in this study. These cell lines were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Gaithersburg, MD) at 37 °C and 5% CO2. For the TF-1 cell line, 2 ng/ml recombinant human GM-CSF was added to the culture. Transfection was performed using the Lipofectamine 2000 transfection reagent (Invitrogen), following the manufacturer's recommended protocol. Briefly, 2 × 105 cells/well (in a 6-well plate) or 6 × 103 cells/well (in a 96-well plate) were transfected with various combinations of the TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL– JAK2(JH2-1), TEL–JAK3(JH1), TEL–JAK3(JH2-1), STAT5b, pTAluc, STAT5 RE(1)/pTA-luc, STAT5 RE(2)/pTA-luc, and STAT5
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RE(4)/pTA-luc plasmids. Twenty-four hours after transfection, protein tyrosine kinase inhibitors (CP-690,550, U0126, or AG490) were added at various concentrations, and the cells were incubated for an additional 24 h. Cell lysates were prepared and analyzed using a luciferase activity assay and western blot. Luciferase activity was assayed using a kit (Promega) and a Victor3 plate reader (Perkin Elmer) and normalized based on β-galactosidase activity. 2.4. Western blot Western blot was performed using standard protocols. Briefly, 48 h after transfection, cells were washed in cold PBS and lysed in RIPA buffer (0.5% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride) containing a protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). After incubation in ice for 1 h, the supernatants were collected by centrifugation at 14,000 ×g for 5 min at 4 °C. The protein concentration was determined using BCA reagents (Pierce, Rockford, IL). A total of 10 μg of protein was resolved using 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) using electroblot. The membrane was probed with the specific primary and secondary antibodies. The polyclonal rabbit antiJAK1, -JAK2, -JAK3, -STAT5b, or -phospho-STAT5b (Tyr694) antibodies (Cell Signaling Technology, Danvers, MA) and mouse monoclonal anti-phosphotyrosine (Cell Signaling Technology) and anti-β-actin antibodies (Sigma) were used as primary antibodies. A horseradish peroxidase-conjugated goat anti-rabbit (Jackson Immunoresearch, West Grove, PA) or anti-mouse (Chemicon, Temecula, CA) IgG antibody were used as secondary antibodies. The signal was visualized using an enhanced chemiluminescence kit (Amersham, Piscataway, NJ). 2.5. Protein kinase inhibitors U0126 [1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] and AG490 (α-Cyano-(3,4-dihydroxy)N-benzylcinnamide) were purchased from Merck Bioscience (Darmstadt, Germany). 2.6. Synthesis of CP-690,550 CP-690,550 was synthesized through the coupling of (Nbenzyl-4-methyl-piperidin-3-yl)methylamine and 4-chloropyrrolo[2,3-d]pyrimidine in the presence of potassium carbonate, followed by hydrogenolytic debenzylation and subsequent cyanoacetamide formation using cyanoacetic acid 2,5-dioxopyrrolidine-1-yl ester. (N-benzyl-4-methyl-piperidin-3-yl)methylamine was prepared from 4-picolin following the literature preparation. 4-Picolin was quarternized with benzyl chloride to produce a pyridinium salt that was reduced with sodium borohydride. The tetrahydropicolin compound was then subjected to a sequence of hydroboration–oxidation, Swern's oxidation, and reductive amination with methylamine. Resolution of the amine with a tartaric acid derivative resulted in enatiomerically pure (N-benzyl-4methyl-piperidin-3-yl)methylamine.
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3. Results 3.1. TEL–JAK fusion protein construction Cytokine-independent JAK activation was induced by constructing TEL–JAK fusion proteins. The oligomerization domain of TEL (amino acids 1–336 of human TEL, Fig. 2A) and the protein tyrosine kinase domain (JH1) and/or pseudokinase domain (JH2) of different JAKs (Fig. 2A) were fused, producing two different constructs for each JAK [TEL–JAK1 (JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2(JH2-1), TEL–JAK3(JH1), and TEL–JAK3(JH2-1)]. TEL–JAK expression constructs were established by subcloning the TEL–JAK (JH1 or JH2-1) fusions into the pcDNA3.1(+) expression vector (Fig. 2B). Fusion protein expression and JAK phosphorylation were confirmed by transfecting the fusion constructs into CV1 cells (monkey kidney fibroblasts) and performing a western blot on extracts from the transfected cells with an anti-JAK antibody and an anti-phosphotyrosine antibody. The 78.6kDa TEL–JAK3(JH1) fusion protein and the 110.6-kDa TEL– JAK3(JH2-1) fusion protein, both with phosphorylated tyrosine residues, were identified (Fig. 2C). The expression and phosphorylation of TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL– JAK2(JH1), and TEL–JAK2(JH2-1) was similarly confirmed using western blot (data not shown). 3.2. STAT5 responsive element reporter system The human STAT5b gene was cloned and inserted into an expression vector (Fig. 3A). The STAT reporter system was generated by cloning 1, 2, or 4 copies of the STAT5 REs (Soldaini et al., 2000), each separated by six nucleotides, into the promoter region of the pTA luciferase vector (RE(1), RE (2), and RE(4), respectively) (Fig. 3B). To evaluate the optimal number of REs in the reporter system, STAT5b and STAT5 RE/pTA-luc were transfected into TF-1, a cytokinedependent erythroblastic cell line. Transfected TF-1 cells were cultured in the presence of 2 ng/ml of GM-CSF for 48 h, and luciferase activity was monitored. Compared with the control, which was transfected only with STAT5b, five times greater luciferase activity was detected in the cells cotransfected with the STAT5b expression plasmid and STAT5 RE(4)/pTA-luc (Fig. 3C). In contrast, cells transfected with the STAT5b expression plasmid plus STAT5 RE(1)/pTA-luc or STAT5 RE(2)/pTA-luc showed a similar level of luciferase activity compared with the control (Fig. 3C). These results indicate that four STAT5 REs allow for the most efficient luciferase expression in response to the binding of dimerized STAT5b. 3.3. Cell-based, JAK3-specific inhibitor screening system To generate a receptor-independent, JAK-dependent STAT activation system, CV-1 cells were co-transfected with 1) TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2 (JH2-1), TEL–JAK3(JH1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(1), RE(2), or RE(4)/pTA-luc. The transfected cells were cultured for 48 h, and luciferase activity was measured. The expression of the fusion proteins including both the JH2 and JH1 domains of JAK3 showed higher luciferase activity than proteins containing the JH1 domain alone (Fig. 3D).
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Fig. 2. Expression and activation of the TEL–JAK fusion proteins. (A) Human TEL, JAK1, JAK2, and JAK3 gene structure. (B) The TEL–JAK fusion constructs. (C) The expression of the TEL–JAK3 fusion protein (left panel) and the activation of JAK3 in the TEL–JAK3 fusion protein (right panel) in the CV-1 cell line were confirmed using western blot with an anti-JAK3 antibody and an anti-phosphotyrosine antibody, respectively. TEL–JAK3(JH1) fusion protein, 78.6 kDa; TEL–JAK3(JH2-1) fusion protein, 110.6 kDa.
Consistent with the results in TF-1 cells, STAT5 RE(4) showed the greatest signal compared with either STAT5 RE(1) or STAT5 RE(2). Additionally, the luciferase activity in CV-1 cells expressing the TEL–JAK3 fusion protein was much higher than that in TF-1 cells, which require the cytokine-dependent activation of endogenous JAKs. The greatest luciferase signal was observed in CV-1 cells transfected with TEL–JAK3(JH2-1) and STAT5 RE(4), and therefore, we used TEL–JAK(JH2-1) and STAT5 RE(4) in the development of the cell-based, highthroughput system to screen for JAK3-specific inhibitors. Importantly, the endogenous JAKs and STATs did not appear to induce luciferase expression in our system (Fig. 3D).
3.4. Sensitivity and specificity of the cell-based, JAK3-specific inhibitor screening system CP-690,550, a JAK3-specific inhibitor (Changelian et al., 2003), was used to evaluate the sensitivity of the system. CV1 cells were co-transfected with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc and cultured for 48 h. Various concentrations of CP-690,550 (101–107 nM) were added for the final 24 h. The IC50 of CP-690,550 for JAK3 was calculated as 12.4 μM (Fig. 4A). The inhibitory activity of CP-690,550 was not due to cytotoxic effects, as the LD50 of CP-690,550 was 1.93 mM (Fig. 4B). The inhibition of JAK3 and downstream STAT5b phosphorylation by CP-690,550 was also confirmed using western blot. With increasing concentrations of CP690,550, the amount of phosphorylated STAT5b decreased, and at concentrations of CP-690,550 over 10 μM, this signal disappeared (Fig. 4C). CP-690,550 did not affect the expression of the transfected STAT5b and TEL–JAK fusion proteins (Fig. 4C). The CV-1 cells used in the assay expressed little
endogenous JAK3 and only a small amount of STAT5b (Fig. 4C). Phosphorylated STAT5b was hardly detected using western blot in the TEL–JAK3(JH2-1) construct only-group or the STAT5 RE(4)/pTA-luc construct only-transfected group (Fig. 4C), indicating that the endogenous STAT5b in CV-1 cells had little effect on our system. The TEL–JAK3(JH2-1)-STAT5bSTAT5 RE(4)/pTA-luc system and its inhibition by CP-690,550 was confirmed using a luciferase assay (Fig. 4D). The specificity of the system was evaluated using the JAK1 and JAK2 constructs and CP-690,550 (100 μM) in the assay. CV-1 cells were co-transfected with 1) TEL–JAK1(JH2-1), TEL–JAK2(JH2-1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(4)/pTA-luc. The inhibitory activity of CP-690,550 was only observed in the cells transfected with TEL–JAK3 (JH2-1); no inhibition was seen with the JAK2 or JAK1 fusion constructs (Fig. 5A). The specificity of the JAK3 system was further evaluated using U0126, an MEK-1 and MEK-2 protein tyrosine kinase inhibitor (Duncia et al., 1998) and AG490, a JAK2-specific inhibitor (Miyamoto et al., 2001) in the assay. JAK3-specific luciferase activity was not inhibited by U0126 (Fig. 5B) or AG490 (Fig. 5C). Furthermore, 105 nM AG490 showed overt cytotoxicity to CV-1 cells (data not shown).
4. Discussion Even with their severe systemic toxicity, calcineurin inhibitors have been the most widely used compounds to suppress unwanted immune responses, such as in the inhibition of graft rejection and in the treatment of autoimmune disease (Meier-Kriesche et al., 2006; Sam and Leehey, 2000). The introduction of novel immunosuppressants that could replace or reduce the dosage of calcineurin inhibitors
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Fig. 3. Construction of a cell-based, JAK3-specific inhibitor screening system. (A) Structure of the human STAT5b gene. (B) The STAT5 RE/pTA-luc DNA constructs. STAT5 RE(1), RE(2), and RE(4) contained 1, 2, and 4 copies of the STAT5 RE sequence, respectively. (C) The efficacy of the STAT5 RE/pTA-luc constructs with 1, 2, or 4 REs was evaluated by co-transfecting cytokine-dependent TF-1 cells with a STAT5b expression plasmid and either STAT5 RE(1), RE(2), or RE(4). The transfected TF-1 cells were cultured in the presence of 2 ng/ml of GM-CSF, and a luciferase activity assay was performed. (D) The cell-based, JAK3-specific inhibitor screening system was made by co-transfecting CV-1 cells with 1) TEL–JAK1(JH1), TEL–JAK1(JH2-1), TEL–JAK2(JH1), TEL–JAK2(JH2-1), TEL–JAK3(JH1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(1), RE(2), or RE(4)/pTA-luc in a 96-well plate. Each combination was analyzed in triplicate, and the data shown are representative of three independent experiments.
could alleviate the lethal systemic side effects of these drugs and provide a better outcome for the patients. γc chain cytokines have been shown to play a critical role during lymphocyte development, activation, survival, and homeostasis (Schmitt and Zuniga-Pflucker, 2005; Gattinoni et al., 2005; Kaech et al., 2003) and have been implicated in critical aspects of immune regulation such as T cell anergy, memory T cell survival and homeostasis, and regulatory T cell development and function (Grundstrom et al., 2000; Tan et al., 2002; Wan and Flavell, 2006). While the clinical proof of concept for the use of JAK3 inhibitors as a putative immunosuppressant has been promising (Kremer et al., 2009), evaluating whether JAK3 inhibitors also affect the induction of tolerance would be challenging.
A cell-based system is essential in finding specific signaling inhibitors that work in the context of molecular networks that normally exist inside a cell. During cytokine signaling, receptor binding is generally required for both JAK phosphorylation (Chen et al., 1997; Zhao et al., 1995) and STAT phosphorylation (Naeger et al., 1999; Yang et al., 1996). Following expression in CV-1 cells, a cytokine-independent cell line, the TEL fusion protein in our study efficiently promoted receptor-independent JAK phosphorylation for JAK1 and JAK3, as well as JAK2, which has been shown to be fused to TEL in some leukemias (Lacronique et al., 1997). Also our strategy of using direct interactions with JAK and STAT through the kinase and/or pseudokinase domain of the JAKs efficiently induced receptor-independent STAT
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Fig. 4. Sensitivity of the cell-based, JAK3-specific inhibitor screening system. (A) CP-690,550, a JAK3-specific inhibitor, was used to test the sensitivity of the system. CV-1 cells were co-transfected with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc and cultured with various concentrations of CP-690,550 for 48 h. The luciferase activity in the transfected cells was measured and compared with controls that were transfected, but treated with PBS. Each sample was tested in triplicate, and the data shown are representative of three independent experiments. (B) The cytotoxicity of CP-690,550 was tested by co-transfecting CV-1 cells with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc. The transfected cells were cultured in various concentrations of CP-690,550 for 48 h, and the viability was checked using a MTT assay. Each sample was tested in triplicate. (C) The system was validated using western blot. Different combinations of TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc were co-transfected into CV-1 cells, and the cells were cultured in the indicated concentration of CP-690,550 for 48 h. The expression of STAT5b and the TEL–JAK3 fusion protein and the activation of STAT5b were analyzed using anti-STAT5b, anti-JAK3, and anti-phospho-STAT5b antibodies, respectively. (D) Different combinations of TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc were co-transfected into CV-1 cells The transfected cells were cultured with 100 μM CP-690,550 or PBS for 48 h, after which a luciferase activity assay was performed. Each sample was tested in triplicate, and the data shown are representative of three independent experiments.
activation. The relative role of the kinase (JH1) and pseudokinase (JH2) domains in the direct interaction with STAT5s is controversial. In our assay, while the luciferase activity was similar between the cells expressing JAK1(JH1) and JAK1(JH2-1), the activity was 100% and 30% greater in JAK2(JH2-1) compared to JAK2(JH1) and JAK3(JH2-1) compared to JAK3(JH1), respectively. In contrast to a previous report demonstrating the exclusive role of the pseudokinase (JH2) domain in direct STAT5 binding in a cell-free protein binding assay, our result suggests that, in this cell-based system, the JH1 domain played a more dominant role in STAT5 activation, especially for JAK1 and JAK3 (Fujitani et al., 1997). By co-transfecting artificially constructed JAK signaling components into a cytokine-independent cell line, we generated a receptor-independent, cell-based assay system to measure JAK activity (Fig. 1). The IC50 of CP-690,550 for JAK3 (12.4 μM) was comparable with the IC50 of CP-690,550 in the inhibition of anti-CD3 and anti-CD28-mediated activation of Jurkat cells (7.8 μM; measured using IL-2 production) (Changelian et al., 2003). This demonstrates
that our cell-based co-transfection assay system showed similar sensitivity with other cell-based functional assays. The IC50 of CP-690,550 for the JAK1 and JAK2 constructs were 775.1 μM and 214.4 μM, respectively (our unpublished data), and this paralleled, with some differences, the IC50 of CP690,550 measured in a cell-free kinase activity assay system (Changelian et al., 2003). It will be very important to use a cell-based assay system to see the physiological effects of the inhibitors. The CV-1 cells used in our assay system showed minimal endogenous STAT5b and JAK3 expression (Fig. 4C) and little background activity of endogenous JAKs as the transfected STAT5b was not phosphorylated in the absence of the co-transfected TEL fusion protein (Fig. 4C). This very low background activity of the endogenous JAK3 or STAT5b was confirmed using a luciferase assay (Fig. 4D). Therefore, these cells would be ideal for our receptor-independent, cell-based JAK assay system. Our cell-based JAK3-specific inhibitor screening system has several advantages. The cytotoxicity of the drugs was monitored simultaneously, and other factors, such as the bioavailability of drugs inside different subcellular
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compartments and the effect of intracellular environments, were reflected in the assay. Therefore, our assay provides a physiological measurement of the JAK3-inhibiting activity of the compound in the context of the normal cellular milieu. As our system can directly test the JAK1-, JAK2-, or JAK3inhibiting capacity of the chemicals simultaneously, specific JAK1 or JAK2 inhibitors can easily be identified. Importantly, JAK2 inhibitors have recently been focused on as novel antileukemia drugs (Atallah and Verstovsek, 2009) and JAK1/ JAK2 inhibitor screening based on cell lines and primary cells from polycythemia vera patients harboring activating JAK2 mutations was recently reported (Pardanani et al., 2009). In addition, our screening method is simple and requires no exogenous cytokines or endogenous receptors, which makes it easily automatable in 96-well or 384-well formats. A virtual screening method to identify novel JAK3 inhibitors was recently reported and this approach may be very useful for pre-screening compounds from the large chemical libraries before performing our cell-based assays (Chen et al., 2008). Acknowledgements This research was supported by grants from MarineBio Technology Project funded by Ministry of Land, Transport and Maritime Affairs and from Korea Science and Engineering Foundation funded by the Ministry of Science and Technology (No. M1064152000106N415200110) and from Korea Healthcare technology R&D Project funded by Ministry for Health, Welfare & Family Affairs (No. A084022). References
Fig. 5. Specificity of the cell-based, JAK3-specific inhibitor screening system. (A) CV-1 cells were co-transfected with 1) TEL–JAK1(JH2-1), TEL–JAK2(JH2-1), or TEL–JAK3(JH2-1); 2) STAT5b; and 3) STAT5 RE(4)/ pTA-luc and cultured with 100 μM CP-690,550 or PBS for 48 h. A luciferase activity assay was then performed. Each sample was tested in triplicate, and the data shown are representative of three independent experiments. (B–C) CV-1 cells were co-transfected with TEL–JAK3(JH2-1), STAT5b, and STAT5 RE(4)/pTA-luc and cultured with various concentrations of (B) U0126 (a MEK inhibitor) or (C) AG490 (a JAK2 specific inhibitor) for 48 h. Luciferase activity assay was then performed. The luciferase activities were compared with the transfected, PBS-treated controls. Each sample was analyzed in triplicate.
Atallah, E., Verstovsek, S., 2009. Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms. Expert Rev. Anticancer Ther. 9, 663. Borie, D.C., O'Shea, J.J., Changelian, P.S., 2004. JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants. Trends Mol. Med. 10, 532. Borie, D.C., Changelian, P.S., Larson, M.J., Si, M.S., Paniagua, R., Higgins, J.P., Holm, B., Campbell, A., Lau, M., Zhang, S., Flores, M.G., Rousvoal, G., Hawkins, J., Ball, D.A., Kudlacz, E.M., Brissette, W.H., Elliott, E.A., Reitz, B.A., Morris, R.E., 2005. Immunosuppression by the JAK3 inhibitor CP-690550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates. Transplantation 79, 791. Changelian, P.S., Flanagan, M.E., Ball, D.J., Kent, C.R., Magnuson, K.S., Martin, W.H., Rizzuti, B.J., Sawyer, P.S., Perry, B.D., Brissette, W.H., McCurdy, S.P., Kudlacz, E.M., Conklyn, M.J., Elliott, E.A., Koslov, E.R., Fisher, M.B., Strelevitz, T.J., Yoon, K., Whipple, D.A., Sun, J., Munchhof, M.J., Doty, J.L., Casavant, J.M., Blumenkopf, T.A., Hines, M., Brown, M.F., Lillie, B.M., Subramanyam, C., Shang-Poa, C., Milici, A.J., Beckius, G.E., Moyer, J.D., Su, C., Woodworth, T.G., Gaweco, A.S., Beals, C.R., Littman, B.H., Fisher, D.A., Smith, J.F., Zagouras, P., Magna, H.A., Saltarelli, M.J., Johnson, K.S., Nelms, L.F., Des Etages, S.G., Hayes, L.S., Kawabata, T.T., Finco-Kent, D., Baker, D.L., Larson, M., Si, M.S., Paniagua, R., Higgins, J., Holm, B., Reitz, B., Zhou, Y.J., Morris, R.E., O'Shea, J.J., Borie, D.C., 2003. Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor. Science 302, 875. Chen, M., Cheng, A., Chen, Y.Q., Hymel, A., Hanson, E.P., Kimmel, L., Minami, Y., Taniguchi, T., Changelian, P.S., O'Shea, J.J., 1997. The amino terminus of JAK3 is necessary and sufficient for binding to the common gamma chain and confers the ability to transmit interleukin 2-mediated signals. Proc. Natl. Acad. Sci. U S A. 94, 6910. Chen, X., Wilson, L.J., Malaviya, R., Argentieri, R.L., Yang, S.M., 2008. Virtual screening to successfully identify novel Janus kinase 3 inhibitors: a sequential focused screening approach. J. Med. Chem. 51, 7015. Duncia, J.V., Santella 3rd., J.B., Higley, C.A., Pitts, W.J., Wityak, J., Frietze, W.E., Rankin, F.W., Sun, J.H., Earl, R.A., Tabaka, A.C., Teleha, C.A., Blom, K.F., Favata, M.F., Manos, E.J., Daulerio, A.J., Stradley, D.A., Horiuchi, K., Copeland, R.A., Scherle, P.A., Trzaskos, J.M., Magolda, R.L., Trainor, G.L., Wexler, R.R., Hobbs, F.W., Olson, R.E., 1998. MEK inhibitors: the
52
K. Oh et al. / Journal of Immunological Methods 354 (2010) 45–52
chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorg. Med. Chem. Lett. 8, 2839. Fujitani, Y., Hibi, M., Fukada, T., Takahashi-Tezuka, M., Yoshida, H., Yamaguchi, T., Sugiyama, K., Yamanaka, Y., Nakajima, K., Hirano, T., 1997. An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT. Oncogene 14, 751. Gattinoni, L., Finkelstein, S.E., Klebanoff, C.A., Antony, P.A., Palmer, D.C., Spiess, P.J., Hwang, L.N., Yu, Z., Wrzesinski, C., Heimann, D.M., Surh, C.D., Rosenberg, S.A., Restifo, N.P., 2005. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J. Exp. Med. 202, 907. Grundstrom, S., Dohlsten, M., Sundstedt, A., 2000. IL-2 unresponsiveness in anergic CD4+ T cells is due to defective signaling through the common gamma-chain of the IL-2 receptor. J. Immunol. 164, 1175. Heissmeyer, V., Macian, F., Im, S.H., Varma, R., Feske, S., Venuprasad, K., Gu, H., Liu, Y.C., Dustin, M.L., Rao, A., 2004. Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat. Immunol. 5, 255. Kaech, S.M., Tan, J.T., Wherry, E.J., Konieczny, B.T., Surh, C.D., Ahmed, R., 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4, 1191. Kremer, J.M., Bloom, B.J., Breedveld, F.C., Coombs, J.H., Fletcher, M.P., Gruben, D., Krishnaswami, S., Burgos-Vargas, R., Wilkinson, B., Zerbini, C.A., Zwillich, S.H., 2009. The safety and efficacy of a JAK inhibitor in patients with active rheumatoid arthritis: results of a double-blind, placebocontrolled phase IIA trial of three dosage levels of CP-690550 versus placebo. Arthritis Rheum. 60, 1895. Lacronique, V., Boureux, A., Valle, V.D., Poirel, H., Quang, C.T., Mauchauffe, M., Berthou, C., Lessard, M., Berger, R., Ghysdael, J., Bernard, O.A., 1997. A TEL–JAK2 fusion protein with constitutive kinase activity in human leukemia. Science 278, 1309. Lacronique, V., Boureux, A., Monni, R., Dumon, S., Mauchauffe, M., Mayeux, P., Gouilleux, F., Berger, R., Gisselbrecht, S., Ghysdael, J., Bernard, O.A., 2000. Transforming properties of chimeric TEL–JAK proteins in Ba/F3 cells. Blood 95, 2076. Leonard, W.J., O'Shea, J.J., 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16, 293. Liptak, P., Ivanyi, B., 2006. Primer: histopathology of calcineurin-inhibitor toxicity in renal allografts. Nat. Clin. Pract. Nephrol. 2, 398. Meier-Kriesche, H.U., Li, S., Gruessner, R.W., Fung, J.J., Bustami, R.T., Barr, M.L., Leichtman, A.B., 2006. Immunosuppression: evolution in practice and trends, 1994–2004. Am. J. Transplant. 6, 1111. Miyamoto, N., Sugita, K., Goi, K., Inukai, T., Lijima, K., Tezuka, T., Kojika, S., Nakamura, M., Kagami, K., Nakazawa, S., 2001. The JAK2 inhibitor AG490
predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome. Leukemia 15, 1758. Naeger, L.K., McKinney, J., Salvekar, A., Hoey, T., 1999. Identification of a STAT4 binding site in the interleukin-12 receptor required for signaling. J. Biol. Chem. 274, 1875. O'Shea, J.J., Pesu, M., Borie, D.C., Changelian, P.S., 2004. A new modality for immunosuppression: targeting the JAK/STAT pathway. Nat. Rev. Drug Discov. 3, 555. Pardanani, A., Lasho, T., Smith, G., Burns, C.J., Fantino, E., Tefferi, A., 2009. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients. Leukemia 23, 1441. Rochman, Y., Spolski, R., Leonard, W.J., 2009. New insights into the regulation of T cells by gamma(c) family cytokines. Nat. Rev. Immunol. 9, 480. Sam, R., Leehey, D.J., 2000. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N. Engl. J. Med. 342, 1837. Schmitt, T.M., Zuniga-Pflucker, J.C., 2005. Thymus-derived signals regulate early T-cell development. Crit. Rev. Immunol. 25, 141. Soldaini, E., John, S., Moro, S., Bollenbacher, J., Schindler, U., Leonard, W.J., 2000. DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites. Mol. Cell. Biol. 20, 389. Stepkowski, S.M., Erwin-Cohen, R.A., Behbod, F., Wang, M.E., Qu, X., Tejpal, N., Nagy, Z.S., Kahan, B.D., Kirken, R.A., 2002. Selective inhibitor of Janus tyrosine kinase 3, PNU156804, prolongs allograft survival and acts synergistically with cyclosporine but additively with rapamycin. Blood 99, 680. Tan, J.T., Ernst, B., Kieper, W.C., LeRoy, E., Sprent, J., Surh, C.D., 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med. 195, 1523. Wan, Y.Y., Flavell, R.A., 2006. The roles for cytokines in the generation and maintenance of regulatory T cells. Immunol. Rev. 212, 114. Yang, C.H., Shi, W., Basu, L., Murti, A., Constantinescu, S.N., Blatt, L., Croze, E., Mullersman, J.E., Pfeffer, L.M., 1996. Direct association of STAT3 with the IFNAR-1 chain of the human type I interferon receptor. J. Biol. Chem. 271, 8057. Zhao, Y., Wagner, F., Frank, S.J., Kraft, A.S., 1995. The amino-terminal portion of the JAK2 protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colony-stimulating factor receptor beta c chain. J. Biol. Chem. 270, 13814.