Getting Syk: Spleen Tyrosine Kinase as a Therapeutic Target

Abstract

Syk is a cytoplasmic protein-tyrosine kinase well known for its ability to couple immune cell receptors to intracellular signaling pathways that regulate cellular responses to extracellular antigens and antigen-immunoglobulin complexes of particular importance to the initiation of inflammatory responses. Thus, Syk is an attractive target for therapeutic kinase inhibitors designed to ameliorate symptoms and consequences of acute and chronic inflammation. Its more recently recognized role as a promoter of cell survival in numerous cancer cell types ranging from leukemia to retinoblastoma has attracted considerable interest as a target for a new generation of anticancer drugs. This review discusses the biological processes in which Syk participates that have made this kinase such a compelling drug target.

Spleen tyrosine kinase

The central role played by Syk (spleen tyrosine kinase) in the immune system in mediating inflammatory responses, coupled with its more recently identified association with malignancy, has made this kinase a popular target for the development of therapeutic agents. A search of the patent literature reveals over 70 filings describing the development of small molecular inhibitors of Syk for the treatment of multiple disease states ranging from arthritis and asthma to leukemia and lymphoma.

Syk catalyzes the phosphorylation of proteins on tyrosines located at sites rich with acidic amino acids [1]. Interestingly, at least two substrates, CD79a [2] and the Ikaros transcription factor [3], have been reported to be phosphorylated by Syk on serine, suggesting that Syk may access a wider distribution of substrates than originally thought. The Syk protein comprises a pair of Src homology 2 (SH2) domains at the N-terminus that are joined to each other by linker A and are separated by a longer linker B from the catalytic domain [4, 5] (Figure 1). Aromatic amino acids in linker A, linker B, the catalytic domain and the extreme C-terminus participate in the formation of a “linker-kinase sandwich” (as first identified in the Syk-family member, Zap-70 [6]) that maintains the enzyme in an autoinhibited conformation [7]. Activation of Syk occurs when the tandem SH2 domains are engaged or when tyrosines participating in the linker-kinase sandwich become phosphorylated.

Figure 1.

Domain organization of Syk. Syk contains an N-terminal, tandem pair of SH2 domains connected by linker A and separated by a linker B region from the catalytic (kinase) domain. Aromatic residues present in linker A, linker B (tyrosines 348 and 352; blue circles), and the catalytic domain interact to form a “linker-kinase sandwich” (dotted circle) that stabilizes the inactive form of the kinase.

SH2 domains are structural motifs that bind phosphotyrosine to promote protein-protein interactions [8]. Each of the SH2 domains of Syk binds proteins containing the sequence pYXXL/I, where pY is phosphotyrosine and X denotes any amino acid. These SH2 domains are juxtaposed in an orientation that allows them to engage simultaneously a linear sequence that contains two of these pYXXL/I cassettes separated by 6-10 amino acids [9]. These high affinity Syk-binding sites are known as immunoreceptor tyrosine-based activation motifs or ITAMs as they are present in many receptors that are important in immune cells [10]. In general, these receptors oligomerize upon engagement by ligand. This is followed by phosphorylation of the two ITAM tyrosines in a reaction that is initiated by a member of the Src-family of tyrosine kinases [4, 5]. Syk physically docks to the doubly phosphorylated ITAM via its tandem SH2 domains in a head-to-tail orientation. Conformational changes disrupt the “linker-kinase sandwich” and activate the enzyme [7]. Syk is then rapidly phosphorylated on tyrosines located in linkers A and B, the activation loop of the catalytic domain, and the extreme C-terminus through both autophosphorylation and phosphorylation in trans by Src-family kinases [4, 5]. These phosphotyrosines serve a variety of purposes including maintenance of the activated state, promotion of signaling complex formation, and release of kinase from the receptor [4, 5]. Signals are further transmitted from the Syk-receptor complex through the phosphorylation of adapter proteins such as BLNK/SLP-65, SLP-76, and LAT [5, 11] (Figure 2). When phosphorylated, these proteins serve as scaffolds to which effectors dock with SH2 or other related phosphotyrosine-binding motifs. Effectors include members of the Tec-family of tyrosine kinases, lipid kinases, phospholipases, and guanine nucleotide exchange factors that further propagate the signal allowing for the activation of multiple pathways including PI3K/Akt, Ras/ERK, PLCγ/NFAT, Vav-1/Rac and IKK/NFκB [4, 5].

Figure 2.

Syk couples FcεRI, the high affinity receptor for IgE, to degranulation in mast cells. Following aggregation of FcεRI by IgE-antigen complexes (not pictured), Lyn initiates the phosphorylation of ITAM tyrosines leading to the recruitment of Syk to the receptor in an interaction mediated by its tandem pair of SH2 domains. Syk becomes phosphorylated in trans by Lyn and by other Syk molecules recruited to the clustered receptor. Active Syk phosphorylates adaptor proteins LAT and then SLP-76, recruited to LAT via GADS (G), to generate binding sites for PLCγ and Btk (not pictured). The phosphorylation of PLCγ by Btk and Syk leads to its activation and the hydrolysis of phosphoinositide 4,5-bisphosphate (PIP₂) to generate the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). The binding of IP₃ to IP₃ receptors on the ER triggers the release of calcium from intracellular stores leading to the entry of extracellular calcium to trigger the release of inflammatory mediators stored in intracellular granules.

It is the nature and function of the receptors in the immune system with which Syk interacts that make it a compelling drug target. Notably, Syk often associates with receptors that bind substances that are foreign to the body (e.g., pathogens or allergens) or that bind antigen- immunoglobulin complexes [5, 10, 12]. Thus, these receptors are prominent among those responsible for discriminating between self and non-self, the sine qua non of the immune system. Unfortunately, when these receptors inappropriately recognize self antigens or harmless environmental antigens, damaging hypersensitivity reactions can result leading to tissue damage and disease.

High affinity receptor for immunoglobulin E (IgE)

Type I hypersensitivity reactions occur when environmental antigens bind to IgE to activate mast cells and basophils to release inflammatory mediators [13]. IgE is produced when dendritic cells that have encountered allergens present peptides on MHC class II molecules to activate naïve CD4⁺ T cells. These helper T cells support the proliferation of allergen-recognizing B cells and secrete cytokines that promote class switching, resulting in the production of IgE. The Fc region of IgE is bound directly by the α-chain of the mast cell receptor FcεRI with high affinity (Kd = 0.1 nM) via an interaction characterized by an exceptionally slow off-rate driven by conformational changes in the bound immunoglobulin [14]. Consequently, IgE is pre-bound to receptors even in the absence of cognate antigen. Mast cells even extend processes into the vasculature to “fish” for circulating IgE [15]. The binding of allergen to the preformed IgE-FcεRI complex clusters the receptor, initiating the phosphorylation by Lyn of ITAM tyrosines in the cytoplasmic tails of the β- and γ-chains of the FcεRI complex. This results in the recruitment and activation of Syk [16]. Syk phosphorylates adaptors including LAT and SLP-76 to recruit both Btk and phospholipase C-γ leading to calcium mobilization and the immediate release of pre-packaged inflammatory mediators (Figure 2). Syk-dependent activation of PKC and the Erk pathway activates phospholipase A2 to initiate the biosynthesis of leukotrienes and prostaglandins. The activation of nuclear factor of activated T cells (NFAT) and NF-κB promotes the expression of a wide array of cytokines and chemokines that precipitate the late phases of an immediate hypersensitivity reaction.

Syk is essential for FcεRI-triggered mast cell activation. Syk-deficient mast cells generated from Syk-knockout mice fail to degranulate in response to FcεRI engagement [17]; and signaling can be restored by the re-expression of Syk [18]. Similarly, mast cells from mice in which a floxed Syk gene has been inducibly excised fail to respond to FcεRI clustering as measured by calcium flux or secretion of histamine [19]. Thus, Syk is an attractive target for therapeutic intervention in mast cell-mediated inflammatory diseases. The disease of most interest to the pharmaceutical industry has been allergic asthma. Mast cells are present at elevated levels in the airway epithelia of asthmatic patients [20, 21] and their activation in bronchopulmonary tissues underlies much of the pathology of allergic asthma [20–22]. Several approaches have been conceived for the generation of drugs that target Syk [23, 24]. These fall into two general categories: small molecule inhibitors of catalytic activity and oligonucleotide-based inhibitors of protein expression. Most small molecule Syk inhibitors, with the exception of the substrate-site inhibitor piceatannol, target the ATP-binding site; several have proven efficacious in blocking inflammation in mouse models of asthma [25–27]. Similarly, inhibitors based on both antisense oligonucleotides and small interfering RNAs (siRNAs) designed to inhibit Syk expression have proven effective in animal models of ovalbumin-induced asthma [28, 29]. For both classes of inhibitors, investigators have explored directed delivery to the lung to avoid side effects associated with systemic drug delivery. The most well developed drug candidate, R343 (Figure 3), although promising in Phase I clinical trials, did not achieve expected endpoints in a recent Phase II trial. However, the exploration of Syk inhibitors for the treatment of asthma is still in its infancy and other inhibitors are in the early stages of clinical trials. Indeed, there are many conditions associated with mast cell-mediated inflammation that could benefit from the use of therapeutic Syk inhibitors. In a particularly interesting example, the intranasal delivery of the Syk inhibitor R112 (Figure 3) provided rapid relief from allergic rhinitis in human volunteers exposed to environmental allergens [30].

Figure 3.

Examples of Syk kinase inhibitors. R788/fostamatinib is the produrg form of R406 and is the best characterized of the Syk inhibitors in patient studies. R343 is an inhaled Syk inhibitor designed for the treatment of allergic asthma and R112 an intranasal inhibitor tested for the alleviation of seasonal allergies. P505-15 is an orally available, highly selective Syk inhibitor. R788, R406, R343 and R112 were developed by Rigel Pharmaceuticals and P505-15 by Portola Pharmaceuticals. All are ATP-binding site inhibitors. Piceatannol, a natural stilbene, is a relatively low affinity inhibitor of Syk, but one that binds competitively with the phosphoacceptor substrate.

Receptors for IgG and arthritis

Type II and type III hypersensitivity reactions are mediated by IgG that interacts with bound or soluble antigens, respectively, and are responsible for the inflammation that accompanies many autoimmune diseases. IgG-antigen complexes are recognized by Fcγ receptors on cells of the innate immune system. Three of these, FcγRI, FcγRIIA and FcγRIIIA, are coupled to the activation of Syk through ITAMs present either as part of the Fc-binding component itself (FcγRIIA) or found on the accessory protein FcRγ (FcγRI, FcγRIIIA) [31]. Receptor engagement triggers the phagocytosis of IgG-opsonized particles and the generation of nitric oxide and reactive oxygen species, which is useful for the killing of microbes but can cause unnecessary tissue damage when triggered by environmental or self-antigens. Syk-deficient macrophages fail to phagocytose IgG-coated particles and neutrophils from Syk-deficient mice fail to undergo an oxidative burst in response to the engagement of FcγRs [32, 33]. The oxidative burst of neutrophils is largely adhesion dependent due to an additional requirement for integrins, heterodimeric receptors that mediate the adhesion of cells to extracellular matrix proteins. In neutrophils, integrins signal through an association with either FcγR or DAP12, another ITAM-containing accessory protein; and Syk is required for adhesion-dependent activation [34]. This makes Syk an attractive target for anti-inflammatory therapeutics.

IgGs that form complexes with self-antigens are important contributors to the pathology of rheumatoid arthritis (RA) [35], a major disease target of therapeutic Syk inhibitors. Immune complexes become deposited in joints and activate macrophages and neutrophils through Fcγ receptors. The resulting production of reactive oxygen species and the secretion of pro- inflammatory cytokines promote severe joint damage. Engineered mice that lack FcRγ or FcγRIII fail to develop arthritis induced by immunization with collagen [36]. Similarly, the transfer of serum from K/BxN mice, which induces arthritis in recipient mice with wild-type immune systems, fails to do so in mice with reconstituted immune systems that lack Syk [37]. In this model, the elimination of Syk from neutrophils alone is sufficient to block joint inflammation [38]. The direct injection of naked Syk siRNA into joints ameliorates swelling and inflammation in a mouse model of arthritis [29], supporting a direct role for Syk inhibition in alleviating RA pathology.

Strategies for the inhibition of Syk in RA patients by small molecules are similar to those for the treatment of asthma although the route of administration is necessarily different. Consequently, considerable effort has been focused on the development of orally available, small molecule, ATP-competitive inhibitors [39]. Of these, the most extensively explored is fostamatinib (R788), a more soluble pro-drug form of the Syk inhibitor R406 (Figure 3). The administration of fostamatinib to whole animals blocks the IgG-mediated Arthus reaction and inhibits collagen-antibody induced arthritis (CIA) [40, 41]. Additional Syk inhibitors also have shown encouraging activity in animal models of RA [42–44]. Fostamatinib has exhibited a promising ability to decrease joint inflammation in Phase II clinical trials in human patients [45–47], however, phase III trials were reportedly disappointing. Clearly, results using other, perhaps more highly selective, Syk inhibitors are anxiously awaited.

As a side-benefit, Syk inhibitors also have the potential to ameliorate other RA-associated pathologies. Osteoclasts, which cause bone damage in patients with RA, are activated through the binding of receptor activator of NF-κB ligand (RANKL) to its receptor RANK. RANK signaling is attenuated by the downregulation of FcRγ and DAP12, RANKL-mediated osteoclast activation is Syk-dependent [48], and Syk inhibitors reduce bone damage in mouse models of RA [41]. The TNFα-induced activation of synoviocytes, which secrete inflammatory cytokines and degradative enzymes that can cause matrix destruction in patients with RA, also is Syk- dependent [49]. Finally, much of the morbidity associated with RA is due to an increased risk of heart failure secondary to atherosclerosis, an inflammatory disease of the vasculature. Damaged endothelial cells attract multiple immune cell types including macrophages, mast cells and platelets that combine to generate atherosclerotic lesions that contain elevated levels of activated Syk and to which are recruited neutrophils. Syk is required for the recruitment of monoctyes and macrophages to endothelial cells expressing fractalkine (CX3CL1) in response to inflammatory cytokines [50]. Syk also mediates integrin-dependent, slow neutrophil rolling and adherence to inflamed endothelial cells expressing L- or P-selectin. The cytoplasmic tail of L-selectin is associated with P-selectin glycoprotein ligand (PSGL-1) and is coupled to the ITAM-containing proteins DAP12 and FcRγ [51]. In mice deficient in the low density lipoprotein receptor, a high cholesterol diet results in the formation of atherosclerotic plaques containing high levels of activated macrophages. The administration of fostamatinib impairs the migration of inflammatory cells into these plaques and also inhibits M-CSF-induced macrophage differentiation [52].

Other IgG-mediated immune disorders

Other examples of autoimmune diseases based on type I or type II hypersensitivity reactions for which an inhibitor of Syk might prove useful are under active investigation. Systemic lupus erythrematosus (SLE) is characterized by B and T cells that are hyperresponsive to the engagement of antigen receptors, leading to the production of auto-reactive antibodies that form immune complexes that can trigger type III hypersensitivity reactions that damage joints, blood vessels and renal glomeruli [53, 54]. The B cells that produce IgG are activated through the B cell receptor for antigen (BCR), which consists of a membrane spanning immunoglobulin in association with two signaling adaptors: CD79a (Ig-α) and CD79b (Ig-β), each of which contains a single ITAM [4, 5]. B cells fail to develop in Syk-deficient immune systems due to an absence of signaling by either pre-B cell receptors or surface IgM on immature B cells resulting in a total loss of the mature B cell population [55]. Similarly, disruption of the Syk gene in DT40 B cells blocks essentially all BCR-stimulated signaling pathways [56]. Thus, B cell activation can be readily blocked by Syk inhibitors. In some patients, B cell hyperactivity is associated with decreased levels of Lyn [57]. Although Lyn is an initiating kinase for BCR signaling, it has a net negative effect on B cell activation mediated by the phosphorylation on tyrosine of inhibitory motifs (ITIMs) on FcγRIIB and CD22, which recruits the SHP-1 and SHIP-1 phosphoprotein and lipid phosphatases. Thus, the selective deletion of Lyn from B cells in mice produces a lupus-like disease [58]. The T cell receptor for antigen (TCR) in mature T cells is associated with the CD3 complex and a dimer of ζ chains, each of which contains three ITAMs [59]. Following receptor engagement, the phosphorylation of ζ chain ITAM tyrosines by Lck leads to the binding of Zap-70 in much the same way that Syk is recruited to phosphorylated ITAMs in B cells or mast cells. Interestingly, in lupus patients, the T cell repertoire is altered such that many cells lack ζ chains, have low levels of Lck, and express Syk, which is not normally found in mature T cells [54]. TCRs in these SLE T cells now signal via the phosphorylation of FcRγ, which replaces the lost ζ chains, and recruits Syk in place of Zap-70. Much of the altered gene expression that characterizes SLE T cells (e.g., increased expression of IL-21, CD44, PP2A and OAS2) can be induced by the overexpression of Syk in normal T cells [60], suggesting that the inappropriate activation of Syk is directly responsible for much of their hyperactive phenotype. Consistent with this hypothesis, Syk inhibitors block TCR-dependent mobilization of calcium in SLE T cells in vitro and fostamatinib inhibits disease progression in murine models of lupus [60–63].

Similarly, inhibitors of Syk are under investigation for the treatment of chronic immune thrombocytopenic purpura (ITP) and heparin-induced thrombocytopenia (HIT), two autoimmune diseases in which self-reactive antibodies are generated against antigens on platelets, resulting in platelet activation and the opsonization and phagocytosis of both platelets and megakaryocytes by macrophages. Syk inhibitors block both immune complex mediated platelet activation through FcγRIIA, and the resulting thrombocytopenia in a mouse model of HIT [64]. Similarly, fostamatinib blocks platelet loss induced by an antibody against integrin αIIβ in a mouse model of ITP [65]. A Phase II clinical trial in human patients showed that fostamatinib successfully restored platelets to approximately 50% of patients with ITP [65].

Type I Diabetes

Immune complexes comprising IgG bound to antigen are internalized by professional antigen presenting cells through Fcγ receptors allowing the presentation of antigen-derived peptides to naïve T cells including CD8 cells in a Syk-dependent process [66]. Thus, in type I diabetes, autoantibodies produced by B cells can facilitate the generation of self-reactive cytotoxic T cells. Deletion of the Syk gene selectively in dendritic cells blocks this generation of diabetogenic T cells [67]. Similarly, in mouse models of type I diabetes, fostamatinib blocks MHC class II- restricted presentation by antigen-specific B cells, the priming of cytotoxic T cells and the onset of diabetes [67]. Thus, Syk inhibitors also have considerable potential for the treatment of autoimmune diseases generally assumed to be mediated predominantly by T cells.

Ischemia-reperfusion injury

The presence of Syk in so many cells that play important roles in inflammatory responses to vascular damage has prompted investigations into the use of kinase inhibitors to treat ischemia- reperfusion injury, which occurs when a blood supply is returned to cells following oxygen and nutrient deprivation [68]. Fostamatinib reduces both intestinal and lung damage in a mesenteric ischemia-reperfusion model [69], potentially by inhibiting FcγR and complement-mediated signaling [70]. Similarly, piceatannol reduces neuronal damage in a mouse model of retinal ischemia-reperfusion injury attributed to the activation of toll-like receptor 4 (TLR4) [71, 72], a receptor with which Syk interacts directly in an unconventional, potentially phosphotyrosine- independent, fashion [73, 73].

Syk and cancer

Several recent studies have indicated an important, albeit enigmatic, role for Syk in tumor cell biology. Syk was first described as a tumor suppressor based on its absence from highly invasive breast carcinomas and its presence in less malignant cells [74, 75]. The ectopic expression of Syk in highly invasive carcinomas inhibits their motility and ability to form metastases [74]. A progressive loss of Syk mRNA is seen in patient samples as cells advance from normal through hyperplastic to ductal carcinoma in situ (DCIS) and then on to infiltrating ductal carcinoma (IDC) [76]. Loss of Syk from breast cancer cells has been attributed to promoter methylation [77] or to allelic loss as recently observed in patient samples of IDC and DCIS [78]. Although the level of Syk expression alone is not prognostic for breast cancer survival, a signature set of Syk “interacting” genes effectively predicts patient outcomes [78]. Similarly, Syk is found in melanocytes, but is frequently absent from melanoma cell lines and tumors due to promoter methylation; and its re-expression induces senescence [79]. Decreases in Syk levels have been reported also in hepatocellular carcinoma at the level of both gene and protein expression [80, 81]. Syk also has reported tumor suppressor activity in pancreatic ductal adenocarcinoma [82].

Of greater interest to drug developers, however, is the increasingly large number of examples in which Syk functions as a pro-survival factor in cancers of both hematopoietic and epithelial origins. Subsets of chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, non- Hodgkin lymphoma and EBV-associated B cell lymphomas express active Syk, the knockdown or inhibition of which leads to apoptosis [83–88]. The general view is that Syk plays its pro- survival role in B cell cancers due to tonic signaling through the BCR [89]. This mechanism may parallel the role of Syk during normal B cell development, where its expression is required for cells to survive the pro- to pre-B and immature to mature B cell transitions, steps that require functional BCRs [90]. However, the repertoire of cells in which Syk functions as a pro-survival factor extends to hematological malignancies not of B cell origin (acute myeloid leukemia (AML) and a variety of T-cell lymphomas [91, 92]) in which it is likely that other ITAM- containing receptors other than the BCR are coupled to the activation of Syk. For example, in AML, a β₃-integrin was identified as an essential gene for leukemia growth whose function is dependent on its downstream target, Syk [93]. Encouragingly, a Phase I/II clinical trial of fostamatinib revealed positive responses for patients with diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma and small lymphocytic leukemia/chronic lymphocytic leukemia [94]. Cells from CLL patients undergoing treatment exhibited decreased expression of BCR target genes and decreased phosphorylation of Btk, a tyrosine kinase activated downstream of Syk [95].

Interestingly, Syk also is a pro-survival factor for some malignancies of epithelial origin where signaling through receptors with ITAMs is more poorly understood. As examples, the expression of Syk distinguishes K-Ras "addicted" lung and pancreatic carcinomas from those not dependent on activated K-Ras for viability [96]. K-Ras addicted cells undergo apoptosis in response to Syk inhibition or knockdown. The expression of Syk is induced by epigenetic mechanisms in retinoblastoma and these cells also undergo apoptosis if the level or activity of the kinase is reduced [97]. The transformation of breast epithelial cells by MMTV requires Syk; but in this case an ITAM is furnished by the MMTV Env protein [98]. Regulation of the alternative splicing of SYK transcripts by EGF promotes expression of the long form of the kinase, which supports the survival of breast and ovarian cancer cells [99]. The downstream signaling events modulated by the presence of a tonically activated Syk are still under investigation, but activation of the PI3K/Akt pathway is a likely contributor (Figure 4). In B cell lymphomas and AML, the activation of mTOR, which lies downstream of Akt, is correlated with the presence of activated Syk [100, 101]. In both retinoblastoma and B-CLL, the level of the anti-apoptotic protein, Mcl-1, is elevated in Syk-expressing cells due to activation of PKCδ and the PI3K/Akt pathway [85, 97, 102]. Active Akt negatively regulates GSK3 to block the phosphorylation of Mcl-1, an event that triggers its ubiquitination and proteosomal degradation [103]. In diffuse large B cell lymphoma (DLBCL), Syk and Akt also repress the expression of the pro-apoptotic protein HRK and upregulate cholesterol biosynthesis, which promotes BCR signaling in lipid rafts [104]. Syk also enhances signaling through the FLT-3 receptor tyrosine kinase in AML cells, many of which express mutant, dominantly active forms of FLT-3 [105].

Figure 4.

Syk couples the BCR to the activation of mTOR and stabilization of the antiapoptotic protein Mcl-1. Binding of antigen to the surface IgM (sIgM) component of the BCR complex leads to the phosphorylation of ITAM tyrosines on the cytoplasmic tails of Igα (CD79a) and Igβ (CD79b) and the recruitment of Syk. Activated phosphorylates adaptor proteins such as CD19 and BCAP to create docking sites for the SH2 domain of the p85 subunit of phosphoinositide 3-kinase (PI3K), which is associated with the p110 catalytic subunit. PI3K catalyzes the formation within the plasma membrane of phosphoinositide 3,4,5-trisphosphate (PIP₃), a ligand for the plexkstrin homology (PH) domain of Akt. Activated Akt catalyzes the phosphorylation of and inhibition of the tuberous sclerosis complex, a negative regulator of mammalian target of rapamycin (mTOR). Akt also phosphorylates and inhibits glycogen synthase kinase 3 (GSK3), blocking the phosphorylation of Mcl-1 and thus enhancing its stability.

Concluding remarks

No Syk inhibitors are currently approved for therapeutic use and recent clinical failures have dampened enthusiasm for the targeting of Syk, at least for the treatment of inflammatory diseases. However, it is not yet clear if treatment failures were a consequence of the target selected (i.e., Syk) or instead derived from a lack of potency or selectivity of the inhibitors that were tested. An analysis of kinase inhibitor selectivity based on competitive binding at the ATP site of a library of kinases indicated that R406, the active metabolite of the most thoroughly tested inhibitor, fostamatinib, was highly promiscuous, binding at a concentration of 3 µM to over 60% of the kinases in the screen (and binding more tightly many kinases than to its intended target) [106]. Consequently, many recent patent filings have focused on the identification of inhibitors with both increased potency and increased selectivity. The true value of Syk as a target for immune disorders will need to wait for further testing of these more potent and specific inhibitors. Other new approaches under development include the design of inhibitors that target simultaneously two kinases (e.g., small molecules capable of inhibiting both Syk and JAK are under investigation for the treatment of hematological malignancies and inflammatory diseases such as chronic dry eye) and the use of Syk inhibitors in combination with other therapeutic agents. In is known, for example, that tumors induced in mice by the expression of active FLT-3 are much more sensitive to a combination of FLT-3 and Syk inhibitors than to either alone [105]. Synergistic growth inhibition also is observed in CLL cells upon treatment with a combination of PI3K and Syk inhibitors [107]). Finally, Syk inhibitor P505-15 attenuates the growth of non- Hodgkin lymphoma and CLL in mouse xenograft models in a manner that is synergistic with fludarabine, a purine nucleoside analog [108]. Finally, it will be important to sort out the mechanisms that govern the apparent dichotomous role of Syk in tumorigenesis where it acts in some cells as a tumor promoter and in others as a tumor suppressor to determine if prolonged administration of Syk inhibitors to patients with chronic inflammatory diseases will ameliorate or exacerbate the metastatic behavior of primary malignancies.

Highlights.

• Syk couples many immune recognition receptors to inflammatory responses

• Syk is a pro-survival factor for several hematological and non-hematological cancers

• Small molecule inhibitors of Syk are under development to treat both immune disorders and cancer