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Key Points Although immunosuppressive therapy is effective at present, toxicity remains an important problem. Many of the existing immunosuppressive agents are directed against ubiquitous targets and therefore have side effects that are unrelated to immunosuppression. Consequently, generating drugs against molecules with restricted expression and/or function might be advantageous. The Janus kinase JAK3 is crucial for signalling by key immunoregulatory cytokines, but has restricted expression and function. This is best illustrated by patients with mutations of the gene encoding this kinase: such children have severe combined immunodeficiency but do not have abnormalities outside of the immune system. This phenotype suggests that JAK3 might be an ideal target. A selective JAK3 inhibitor, CP-690,550, has now been generated to effectively block immune responses both in vitro and in vivo . Other JAK3 inhibitors have been previously described, but none are as potent or selective as CP-690,550. This drug is effective in models of transplant rejection and is not associated with the toxicities that are seen with other immunosuppressive agents. A JAK3 inhibitor is likely to have uses in many settings beyond transplantation, including autoimmune disease and possibly haematopoietic malignancy. Targeting other JAKS and other elements in the JAK/STAT pathway is also conceptually appealing. On the basis of the phenotype that is associated with TYK2 deficiency, a TYK2 antagonist might be useful in inhibiting diseases that are characterized by the activation of T H 1 cells. Given their importance in malignant transformation and immunoregulation, STAT proteins have received considerable attention as therapeutic targets and STAT inhibitors are being studied at present. SOCs proteins are cytokine-induced feedback inhibitors of signalling, which can also be considered as potential targets. Thousands of organs are transplanted each year and millions of people suffer from autoimmune diseases, which creates a need for an armamentarium of immunosuppressive drugs. Unfortunately, immunosuppressants have unwanted side effects owing, in part, to the fact that they have ubiquitous molecular targets. Cytokines have emerged as important controllers of the immune response, and work during the past decade has identified Janus kinases (JAKs) and signal transducers, and activators of transcription (STATs), as crucial intracellular elements in cytokine signalling. Here, we discuss the potential of the JAK/STAT pathway as a target for new immunosuppressants. In particular, the inhibition of JAK3 seems to be an excellent strategy, because of the selective expression and precise functions of this kinase.

JAK3 is a protein tyrosine kinase that specifically associates with the common γ chain (γ c), a shared subunit of receptors for interleukin (IL) 2, 4, 7, 9, and 15. Patients deficient in either JAK3 or γ c presented with virtually identical forms of severe combined immunodeficiency (SCID), underscoring the importance of the JAK3-γ c interaction. Despite the key roles of JAK3 and γ c in lymphocytic development and function, the molecular basis of this interaction remains poorly understood. In this study, we have characterized the regions of JAK3 involved in γ c association. By developing a number of chimeric JAK3-JAK2 constructs, we show that the binding specificity to γ c can be conferred to JAK2 by transferring the N-terminal domains of JAK3. Moreover, those JAK3-JAK2 chimeras capable of binding γ c were also capable of reconstituting IL-2 signaling as measured by inducible phosphorylation of the chimeric JAK3-JAK2 protein, JAK1, the IL-2 receptor β chain, and signal transducer and activator of transcription 5A. Subsequent deletion analyses of JAK3 have identified the N-terminal JH7-6 domains as a minimal region sufficient for γ c association. Furthermore, expression of the mutant containing only the JH7-6 domains effectively competed with full-length JAK3 for binding to γ c. We conclude that the JH7-6 domains of JAK3 are necessary and sufficient for γ c association. These studies offer clues toward a broader understanding of JAK-mediated cytokine signaling and may provide a target for the development of novel therapeutic modalities in immunologically mediated diseases.

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, γ c and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

p59fynT is a protein tyrosine kinase in the src family that has been associated with and believed to function in the signaling of many receptors, including the T-cell receptor. A role for the kinase in antigen-driven pulmonary inflammation was examined using mice whose p59fynT gene had been genetically ablated. FynKO mice that were sensitized to ovalbumin exhibited a marked increase in bronchoalveolar lavage eosinophils and cytokines, including interleukin (IL)-4 and IL-5, relative to wild-type mice in response to antigen aerosol exposure. Ovalbumin-stimulated IL-5 production was also increased in cultured splenocytes derived from fynKO mice relative to wild-type mice, whereas interferon-gamma levels were unchanged. Diminished concanavalin A--stimulated IL-4 levels from fynKO splenocytes were consistent with reduced serum immunoglobulin (Ig)E levels observed in sensitized/saline aerosol-challenged animals and may reflect defective natural killer 1.1(+) T cell development. Normalization of IgE levels in sensitized fynKO mice relative to wild-type mice occurred after repeat antigen challenge, which suggests a secondary source of IL-4. Overall, these data demonstrate fyn is a negative regulator of allergic airway inflammation in mice because its absence promotes a shift to a T helper-2 phenotype that may reflect the kinase's role in T-cell receptor signaling.

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

The NGFI-A gene encodes a ``zinc-finger'' protein that is rapidly induced by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. The complete exon/intron organization and nucleotide sequence of the rat NGFI-A gene have been determined. The gene spans 3789 nucleotides (nt) and is interrupted by a single intron at nt 588. All three zinc-finger DNA-binding domains are contiguously coded for within the 3′ exon; this is in contrast to the structure described by others for the Xenopus laevis transcription factor TFIIIA gene. To analyze the transcription of this gene, we have determined the transcription start site and nucleotide sequence of the 5′ flanking region. Transfection of PC12 cells with a fragment from the 5′ flanking region linked to the chloramphenicol acetyltransferase (CAT) gene revealed that it contains an element which imparts an NGF-inducible phenotype to the normally silent CAT gene. Several regions with homologies to recognizable sequence elements are present in this fragment, including a TATA box at nt -27, serum response elements at nt -84, -106, -370, and -408, a cAMP-responsive element at nt -140, and a transcription factor Sp1-binding site at nt -286. These results establish the genomic structure of this mammalian multifinger protein and demonstrate that an NGF-responsive element lies upstream of the NGFI-A gene.

The Janus kinase (JAK) JAK3 plays a vital role in the intracellular signaling cascade downstream of select cytokine receptors in immune cells. Inhibition of JAK3 is expected to result in potent immunosuppression, and thus JAK3 appears to be a rational pharmaceutical target. Nonclinical and clinical data relating to transplantation have become available for several molecules targeting inhibition of JAK3. Recent studies suggest that some of these agents lack potency and selectivity for JAK3 or the JAK family. Published data on these relatively nonselective agents must, therefore, be interpreted with caution. Clinical trials on CP‐690,550 in calcineurin‐inhibitor‐free regimens have provided preliminary evidence that JAK3 inhibition is a promising approach to prevent acute allograft rejection. However, dose‐limiting adverse events, such as opportunistic viral infections and hematological aberrations, have been observed. Further evaluations of this class of agents are warranted to ascertain the dose/concentration range for individual agents and the combination regimens that optimize clinical efficacy and minimize adverse effects.

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.