Inhibition of PI3K Signaling Spurs New Therapeutic Opportunities in Inflammatory/Autoimmune Diseases and Hematological Malignancies

A. Phosphoinositide 3-Kinase γ and δ Coordinate Leukocyte Migration

There is now strong evidence that PI3Kγ and PI3Kδ act in partnership to regulate immune cell signaling and function. The directed movement of leukocytes from the circulation into tissues or secondary lymphoid organs is essential for routine immunosurveillance and normal host defense during infection or injury. Cell migration was initially predicted to require input from PI3Kγ, given that many of the homeostatic and inflammatory mediators that regulate leukocyte trafficking function via GPCRs. Several studies demonstrated that PI3K inhibitors or genetic loss of PI3Kγ causes reduction in chemotactic responses of lymphocytes, neutrophils, macrophages, and eosinophils in a variety of in vitro and in vivo migration assays (Hirsch, 2000; Sasaki et al., 2000b; Reif et al., 2004; Smith et al., 2007; Lim et al., 2009; Thomas et al., 2009). The catalytic function of PI3Kγ is crucial for morphological changes associated with cell polarization by generating PI(3,4,5)P₃ at the leading edge and regulating Rac activity and the cytoskeleton reorganization (Costa et al., 2007; Ferguson et al., 2007; Barberis et al., 2009). An additional route by which class IA PI3Ks can influence cell migration has recently been uncovered and involves the regulation of transcription factors that regulate cell quiescence and expression of homing receptors on T cells. Hence, genetic and pharmacological studies have revealed that PI3Kδ and PDK1/Akt play an essential role in the events that lead to proteolytic shedding and reduced transcription of CD62L, CCR7 and S1P receptor-1 (Finlay et al., 2009; Waugh et al., 2009). However, PI3Kδ also regulates neutrophil migration as demonstrated by the ability of a PI3Kδ-specific inhibitor to reduce directional neutrophil movement in response to chemotactic agents (Sadhu et al., 2003; Puri et al., 2004). Moreover, several studies have also revealed the importance of endothelial activity of both PI3Kγ and PI3Kδ in regulating neutrophil interactions with the inflamed vessel wall (Puri et al., 2005, 2004).

The evidence discussed above points to partially overlapping roles of both PI3Kγ and PI3Kδ isoforms in regulating the complex interplay between leukocytes and the inflamed endothelium, as well as their activation responses. Indeed, analysis of neutrophil migration in vivo revealed that, in fact, although PI3Kγ is important in early chemokine-induced emigration, PI3Kδ replaces and maintains the delayed chemokine-induced neutrophil recruitment into inflamed tissues (Liu et al., 2007), suggesting a coordinated but temporally distinct role for these isoforms. This is consistent with observations that in tumor necrosis factor-α (TNFα)-primed human neutrophils, fMLP induces a biphasic increase in PI(3,4,5)P₃ accumulation, in which the first phase depends on the PI3Kγ isoform. The second phase of PI(3,4,5)P₃ accumulation depends on prior exposure to TNFα and is driven predominantly by PI3Kδ (Condliffe et al., 2005). There is also evidence for distinct yet complimentary roles for PI3Kγ and PI3Kδ at different stages of transendothelial migration of effector T cells (Jarmin et al., 2008; Ward and Marelli-Berg, 2009) as well as during mast cell degranulation in response to antigen receptor and chemokine signals (Laffargue et al., 2002; Ali et al., 2004, 2008; Willox et al., 2010; Kitaura et al., 2005). Indeed, mice that are both deficient in PI3Kγ and express the kinase-inactive PI3Kδ^D910A mutant (unlike mice in which PI3K genes have been targeted individually) display severe impairment of thymocyte development, profound T-cell lymphopenia, and T-cell and eosinophil infiltration of mucosal organs, elevated IgE levels, and a skewing toward Th2 immune responses (Ji et al., 2007). However, the serious defects in immune development observed in the PI3Kγδ-null mice prevent detailed dissection of the selective roles of these PI3K subunits in post-thymic responses.

Despite evidence for cooperation between these isoforms, it is important to highlight that genetic targeting of both PI3Kγ and -δ has revealed distinct contributions of these isoforms dependent on receptor and/or cell type and activation state. Thus, although both PI3Kγ and -δ are required for migration of neutrophils toward leukotriene B4 (LTB4), only the activity of PI3Kγ is responsible for the migration of cells in response to fMLP (Randis et al., 2008). Likewise, both PI3Kγ and PI3Kδ are necessary for natural killer (NK) cell migration to inflamed tissues and the uterus during early pregnancy in vivo and chemotaxis to CXCL12 and CCL3 in vitro. In contrast, PI3Kδ alone was required for NK cell distribution in steady state as well as for trafficking to lymphomas and for chemotaxis to S1P and CXCL10 in vitro (Saudemont et al., 2009). Other studies in murine models have shown that in vitro, both PI3Kδ and PI3Kγ are required for FcγRI-driven mast cell degranulation; in vivo, PI3Kγ (but not PI3Kδ) is dispensable for allergic responsiveness (Laffargue et al., 2002; Ali et al., 2004, 2008; Kitaura et al., 2005). The changing dependence on either or both of these PI3K isoforms is important to bear in mind when designing drugs to interfere with these isoforms in the immune system.

B. Phosphoinositide 3-Kinase β and δ Coordinate the Respiratory Burst

Once neutrophils encounter bacteria, they generate reactive oxygen species (ROS) as part of the respiratory burst to destroy bacteria and resolve infection. Neutrophils isolated from PI3Kγ-null mice show reduced superoxide generation, but in a model of invasive fungal infection of the lung, neutrophils from PI3Kγ-null mice were similar to wild-type cells in their ability to produce ROS in response to Aspergillus fumigates hyphae (Boyle et al., 2011). In this model, loss of both PI3Kγ and PI3Kδ isoforms partially reduced ROS generation. However, neutrophils from mice lacking PI3Kβ and expressing kinase-dead PI3Kδ^D910A displayed a marked reduction in neutrophil ROS generation. The importance of PI3Kβ and its cooperation with PI3Kδ was further underlined by a study from the same group that demonstrated PI3Kβ plays an essential, nonredundant role in the efficient activation of mouse neutrophils by IgG-containing immune complexes (Kulkarni et al., 2011). Neutrophils detect these immune complexes through Fc receptors for IgG (FcγRs). PI3Kβ was shown to act downstream of FcγR phosphorylation, and PI3Kβ-deficient neutrophils produced lower concentrations of ROS in response to immune complexes than control neutrophils. This effect of PI3Kβ deficiency was most pronounced at low immune-complex densities, but at high densities, PI3Kδ could partly compensate for the loss of PI3Kβ in ROS generation. The study also suggests that LTB4 is produced in response to FcγR ligation and signals in an autocrine or paracrine manner through its GPCR BLT1 to enhance immune complex-induced ROS production. Remarkably, this signaling loop in response to FcγR ligation was not abolished in PI3Kγ-deficient neutrophils, despite the known role for PI3Kγ downstream of BLT1. Rather, although the response to LTB4 alone required PI3Kγ, the response to costimulation with LTB4 and immune complexes was largely dependent on PI3Kβ. Hence, PI3Kβ can integrate signals from FcγRs and BLT1 and plays a crucial role in the response to immune complexes (Kulkarni et al., 2011). This was further illustrated in mice lacking PI3Kβ, which were markedly protected in a model of autoantibody-induced skin blistering. Given that the loss of PI3Kβ does not affect humoral responses or neutrophil-mediated killing of Staphylococcus aureus (Kulkarni et al., 2011), inhibition of PI3Kβ might be an effective option to treat certain inflammatory conditions but avoid general adverse effects on protective immunity.

A. The Pathfinder Inhibitors: The Influence of Crystal Structure Elucidation and Cancer

The first compounds to block PI3K, the natural product wortmannin and Eli Lilly's 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), have served as useful experimental tools with which to explore the PI3K pathway. Wortmannin was first identified nearly 40 years ago (Wiesinger et al., 1974), although its inhibitory effects on PI3K were not identified until the early 1990s (Arcaro and Wymann, 1993), when it was found to covalently interact with the ATP binding pocket of PI3K. LY294002, developed by Lilly Research Laboratories (Indianapolis, IN), was an important first step in the synthesis of compounds to target PI3K (Vlahos et al., 1994). A reversible competitive inhibitor of the ATP binding pocket of PI3K, LY294002 is less potent than wortmannin and has selectivity and toxicity issues (Gharbi et al., 2007). The value of these crude but effective inhibitors in advancing the PI3K field should not be underestimated and is evidenced (at the time of writing) by just over 6000 PubMed hits for LY294002 alone. Indeed, these compounds allowed the initial identification or hint of a role for PI3K in various biological processes.

The usefulness of wortmannin and LY294002, as both research tools and potential drugs, was tempered by their broad targeting of all PI3K isoforms and off-target effects (Gharbi et al., 2007). Despite this, the simpler planar structure of LY294002 has helped the design of many more selective pan-PI3K and isoform-selective inhibitors. In particular, X-ray crystallography data of PI3Kγ bound to wortmannin and LY294002 helped the understanding of the manner by which these compounds fit into the ATP binding pocket (Walker et al., 2000), which in turn influenced attempts to design better compounds with increased potency and required selectivity. Although this was in part fuelled by the desire for better anti-inflammatory drugs, the single most important catalyst for PI3K inhibitor design was arguably the potential application of PI3K inhibitors as anticancer drugs. The PI3K/Akt/mTOR pathway is one of the most commonly activated pathways in human cancer and is central to the transformed phenotype of most cancer cells (Manning and Cantley, 2007; Engelman, 2009). It can be activated by amplification or activating mutation of either PI3Kα or upstream receptor tyrosine kinases, and by mutations or deletions downstream in the pathway or of regulatory elements such as the phosphatase PTEN (Yuan and Cantley, 2008; Liu et al., 2009b). Increased PI3Kα activity or activity-enhancing mutations in the PI3KCA gene are common in a number of cancers, including breast, endometrial, and glioblastoma (Samuels et al., 2004; Kok et al., 2009).

Crystal structures of PI3Ks have revealed six regions within the ATP binding pocket: hydrophobic regions I (the affinity pocket) and II, the hinge region, the P loop, the start of the activation loop (DFG motif), and the specificity pocket (Walker et al., 2000). Designing compounds that explore or create these pockets has improved potency and selectivity of PI3K inhibitors (Knight et al., 2006; Folkes et al., 2008; Berndt et al., 2010). These have influenced subsequent efforts to develop PI3K inhibitors with improved selectivity and reduced toxicity. In addition, mTOR shares high sequence homology in the hinge region with PI3K, and therefore some compounds originally developed as PI3K inhibitors were later shown to also target mTOR. Furthermore, pan-PI3K isoform and mTOR inhibitors often exert synergistic effects in functional measures. Approximately 18 drugs now in clinical development target the PI3K signaling cascade; of these, half target both mTOR and PI3K, most target one or more PI3K isoforms, and some inhibit DNA protein kinase (Table 2).

B. Where Is the Best Site to Block the Phosphoinositide 3-Kinase Signaling Cascade?

The targeting of PI3K in cancer has prompted much debate regarding exactly the optimal point to inhibit in this cascade. The most popular targets to date have been PI3K catalytic isoforms, Akt, and mTOR (Fig. 3). Inhibitors of class I PI3K would prevent accumulation of PI(3,4,5)P₃, which is necessary for full activation of Akt/PKB. However, Akt can be phosphorylated and activated by inhibitor of nuclear factor κB kinase subunit ε (IKBKE) independently of the recognized PI3K/PDK1/mTORC2/PH domain-mediated mechanism and thus, PI3K inhibition may not fully silence Akt/PKB and hence mTORC1 activation (Guo et al., 2011). Direct targeting of Akt has been explored with some success, particularly with respect to cancer (Hers et al., 2011; Tan et al., 2011). This would avoid disrupting effects of PI3K that are mediated by other downstream effectors of PI3K signaling, which may or may not be desirable depending on the disease context. Paradoxically, inhibition of Akt could actually increase PI3K-dependent activation of those effectors, because Akt activation leads to increased mTORC1 activity, which operates a well known negative feedback mechanism that inhibits insulin-stimulated PI3K activation (Guertin and Sabatini, 2009). Because of this negative feedback, when mTORC1 is active, the PI3K-Akt pathway is suppressed, whereas if mTORC1 is inhibited (for instance, with rapamycin), PI3K-Akt signaling can actually be enhanced. Indeed, success in developing effective mTOR inhibitors was tempered by the recognition that currently approved inhibitors of mTORC1 fail to block the activation of Akt via mTORC2 at Ser-473 (Moore et al., 2011). In cancer cells, losing this feedback inhibition may actually promote survival and counter the potential therapeutic benefits of mTORC1. Moreover, success in developing effective mTOR inhibitors was tempered by the recognition that currently approved inhibitors of mTORC1 fail to block the activation of Akt via mTORC2 at Ser-473 (Moore et al., 2011). Thus, inhibition of both mTORC1 and mTORC2 would therefore be predicted to give a better potential for efficacy by removing the mTORC2-mediated phosphorylation of Akt. In this regard, both ATP competitive [e.g., AZD8055 ([5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl]methanol) from AstraZeneca] and allosteric (from Pathway Therapeutics, San Francisco, CA) inhibitors of mTORC1 and mTORC2 have been reported, although no structures have been published for the latter compound. However, the best inhibition of PI3K signaling (at least in the context of anticancer drugs) would be predicted to be achieved by dual targeting PI3K and mTORC1/2, because this would effectively shut down signaling at both proximal and intermediate sites of the cascade (Fig. 3). This would be the case with SF1126 [(8S,14S,17S)-14-(carboxymethyl)-8-(3-guanidinopropyl)-17-(hydroxymethyl)-3,6,9,12,15-pentaoxo-1-(4-(4-oxo-8-phenyl-4H-chromen-2-yl)morpholino-4-ium)-2-oxa-7,10,13,16-tetraazaoctadecan-18-oate; Semaphore Pharmaceuticals, Indianapolis, IN], a conjugate of LY294002 with an RGD-containing peptide. The purpose of the RGD peptide was to increase solubility and binding to specific integrins within the tumor compartment. This results in enhanced delivery of the active compound to the tumor vasculature and tumor while limiting its damaging systemic effects. SF1126 inhibits not only PI3K and mTOR but also a select number other cancer targets such as DNA protein kinase, PIM1, and PLK1 (Gharbi et al., 2007). Hence, SF1126 therefore effectively shuts down PI3K-Akt-mTOR signaling. It has shown promising preclinical results alone or in combination with the anticancer drug doxorubicin and not only decreases proliferation but also has antiangiogenic and pro-apoptotic actions (Garlich et al., 2008; Ozbay et al., 2010; Peirce et al., 2011).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Sites of intervention of PI3K/mTOR-targeting inhibitors. Heterodimeric PI3Kα, PI3Kβ, and PI3Kδ complexes are activated downstream of growth factors, antigen and cytokine receptors, whereas PI3Kγ activation is triggered downstream of GPCRs. PI3K phosphorylates PI(4,5)P₂ to generate PI(3,4,5)P₃. This reaction can be reversed by the action of PTEN (not shown). Alternatively, the 5′ phosphatase SHIP-1 can convert PI(3,4,5)P₃ to PI(3,4)P₂. Akt and PDK1 directly bind to PI(3,4,5)P₃ and are thereby recruited to the plasma membrane. The phosphorylation of Akt at Thr308 (which is in the activation loop of Akt) by PDK1 and at Ser-473 (which is in a hydrophobic motif of Akt) by mTORC2 results in full activation of this protein kinase. In turn, Akt phosphorylates several cellular proteins (not shown) to facilitate cell survival and cell cycle entry. In addition, Akt phosphorylates and inactivates tuberous sclerosis 2 (TSC2), a GTPase-activating protein for Ras homolog enriched in brain (RHEB). Inactivation of TSC2 allows RHEB to accumulate in the GTP-bound state and thereby activate mTORC1. Additional activation of mTORC1 can occur via input from other pathways upon nutrient activation. This figure shows potential sites for pharmacological intervention in the PI3K/Akt/mTOR signaling cascade (green shaded areas). 1, inhibitors of class I PI3K would prevent accumulation of PI(3,4,5)P₃, which is necessary for full activation of Akt/PKB. This approach would not prevent activation of mTORC1 via PKB-independent avenues or mTORC2 activation. 2, Akt can be phosphorylated and activated by IKBKE, independently of the recognized PI3K/PDK1/mTORC2/PH domain-mediated mechanism; thus, PI3K inhibition may not fully silence Akt/PKB and hence mTORC1 activation. 3, mTORC1 inhibitors will alleviate a negative feedback mechanism that targets the PI3K pathway and potentially enhance PI3K-Akt signaling. 4, inhibition of both mTORC1 and mTORC2 would therefore be predicted to give a better potential for efficacy by removing the mTORC2-mediated phosphorylation of Akt. However, the best inhibition of PI3K signaling would be predicted to be achieved by dual targeting PI3K and mTORC1/2, because this would effectively shut down signaling at both proximal and intermediate sites of the cascade. Direct targeting of Akt in particular has been explored with some success but is not depicted here. Targets of inhibitors currently in clinical trial are depicted: those in red represent compounds that are selective for a single PI3K isoform; inhibitors in green exhibit PI3Kγ/δ dual selectivity; inhibitors in orange are mTOR catalytic site inhibitors. Compounds targeting SHIP-1 are allosteric activators. Negative regulatory events are depicted by red lines.

The optimal site for therapeutic intervention in the PI3K signaling cascade may require targeting of one or more components of the signaling cascade and will likely depend on the particular molecular pathology driving a given disease. From an immunological perspective, targeting of mTOR in the immune system, particularly in T cells, has long been the focus for the development of immunosuppressive drugs such as rapamycin and its analogs, which suppress mTOR activity via an allosteric mechanism (Guertin and Sabatini, 2009). We now appreciate that mTOR provides a critical link between metabolic demands and cellular function and plays a role in regulating diverse immune cells, including neutrophils, mast cells, NK cells, γδ T cells, macrophages, dendritic cells, T cells, and B cells (Delgoffe and Powell, 2009; Mills and Jameson, 2009; Thomson et al., 2009; Weichhart and Säemann, 2009). Some of the new mTORC1/mTORC2 inhibitors developed primarily as anticancer agents have been characterized in the context of immune diseases. The mTORC1/2 inhibitor PP242 [(2Z)-2-(4-amino-1-propan-2-yl-2H-pyrazolo[3,4-d]pyrimidin-3-ylidene)indol-5-ol] has potent antileukemic effects in vitro and in vivo but leaves lymphocyte function largely intact (Janes et al., 2010). PP242 has also shown promise in treating multiple myeloma (Hoang et al., 2010), whereas another mTORC1/2 inhibitor, INK128 [3-(2-amino-5-benzoxazolyl)-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine], developed by Intellikine (La Jolla, CA), inhibits cell proliferation both in vitro and in vivo. INK128 has entered phase I clinical trials as a therapeutic agent for the treatment of breast cancer (ClinicalTrials.gov identifier NCT01351350), lymphoma (ClinicalTrials.gov identifier NCT01058707), and multiple myeloma (ClinicalTrials.gov identifier NCT01118689). The dual targeting of PI3K/mTOR in inflammatory/autoimmune disease has not been extensively investigated. Nevertheless, dual pan-PI3K/mTORC1/2 inhibitors seems to cause greater immune suppression than mTORC1/2 alone, with greater reduction of hematopoietic colony formation and B- and T-cell proliferation, a lowered fraction of B cells with germinal center phenotype, as well as percentages of total splenic B and T lymphocytes. Pan-PI3K-TORC1/2 inhibitors also suppress immune rejection of melanoma xenografts (López-Fauqued et al., 2010).

One concern of the dual PI3K/mTOR inhibitor approach is that it is so robust that it might lead to general immunosuppression. This may be acceptable in transplantation settings but possibly not for other diseases, such as inflammatory and autoimmune diseases, where a lighter touch might be more appropriate to dampen the deleterious effects of an overactive immune system rather than silence it completely. The long appreciated important role of PI3Kδ and -γ (as well as a growing understanding of the contribution of PI3Kβ), in both innate and adaptive immunity has therefore led to the search for selective pharmacological tools with which to target these enzymes in inflammation and autoimmune disorders.

A. Phosphoinositide 3-Kinase δ Inhibitors

Given the high degree of similarity that exists between the amino acids forming the ATP-binding pockets of the four class I PI3Ks, it was expected that isoform-selective inhibitors with a reasonable (at least 50-fold) difference in potency would be difficult to obtain. However, the discovery of the quinazolinone purine series, exemplified by the ICOS Corporation (Bothell WA) compound IC-87114 (2-[(6-aminopurin-9-yl)methyl]-5-methyl-3-(2-methylphenyl)quinazolin-4-one), indicated that this task was possible (Sadhu et al., 2003). IC-87114 demonstrated an IC₅₀ value of approximately 100 nM for PI3Kδ lipid kinase activity and was also potent in cell assays that are dependent on the catalytic activity of this isoform but had negligible potency against the PI3Kα and PI3Kβ isoforms (Table 3).

In 2006, several members of ICOS Corporation formed a spin-off company, Calistoga Pharmaceuticals (Seattle, WA). Calistoga developed CAL-101 (5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]quinazolin-4-one), a PI3Kδ-specific inhibitor, which has experienced successful proof of concept in clinical trials for treatment of B-cell malignancies (in section VII.A). This compound (which was acquired by Gilead in February 2011 and recently renamed GS-1101) exhibits 40- to 300-fold selectivity over other PI3K isoforms (Table 3). Calistoga Pharmaceuticals is also exploring inhibition of PI3Kδ in inflammatory conditions using CAL-101 and CAL-263 (Norman, 2011), another PI3Kδ-selective inhibitor (ClinicalTrials.gov identifiers NCT00836914 and NCT01066611). In addition, patents have been filed by several other companies [Amgen (Thousand Oaks, CA), Intellikine, and Incyte Systems (Pal Alto, CA)] describing PI3Kδ inhibitors, and the majority are based on the same basic pharmacophore identified by ICOS (Norman, 2011). However, additional scaffolds have now been reported by several companies; almost all of these are with intended indications against B-cell lymphomas (Norman, 2011).

A better understanding of the molecular mechanisms of isoform selectivity of PI3Kδ inhibitors has been made possible by a recent report describing the crystal structure of the PI3Kδ catalytic core, both free and complexed with a broad panel of new and mostly PI3Kδ-selective PI3K inhibitors. Comparison of the PI3Kδ structure with those of other isoforms reveals several important features for inhibitor design: PI3Kδ is more flexible than PI3Kγ, and most of the PI3Kδ-selective inhibitors adopt a propeller shape that allows them to exploit this conformational plasticity by opening up and then binding into a “specificity pocket.” In contrast, pan-specific PI3K inhibitors adopt a flat structure rather than the propeller shape and do not access the specificity pocket. This new understanding was used to design novel inhibitors that retained very high selectivity toward PI3Kδ by exploiting the selectivity pocket, combined with much increased potency by targeting the affinity pocket. This study provides the first detailed structural insights into the active site of a class IA PI3K occupied by noncovalently bound inhibitors and suggests mechanisms to increase the potency of inhibitors without sacrificing isoform selectivity and also how to optimize solubility, pharmacokinetics/metabolism, and pharmacodynamic behavior (Berndt et al., 2010).

B. Phosphoinositide 3-Kinase γ Inhibitors

Selective inhibition of PI3Kγ has been accomplished in a series of compounds designed by Merck Serono SA based on the thiazolidinedione scaffold. One of these, AS-605240 [5-(quinoxalin-6-ylmethylidene)-1,3-thiazolidine-2,4-dione] has demonstrated superior potency for PI3Kγ compared with related compounds (Table 3), can be administered orally and has high membrane permeability (Barber et al., 2005; Camps et al., 2005). Despite the promising preclinical data using these compounds (described in section VII), PI3Kγ inhibitors have yet to undergo further development or clinical proof of concept. In general terms, the level of selectivity of compounds against PI3Kγ versus other PI3K isoforms has been largely disappointing. Possible reasons for the relatively slow progress in developing PI3Kγ inhibitors include the close structural conservation of class I PI3Ks and other lipid kinases in the ATP-binding pocket and the limited ability of the commonly used in vitro assays based on recombinant enzymes, to predict cellular and in vivo kinase selectivity. Researchers at Cellzome (Heidelberg, Germany) made a recent advance in this area. They developed a chemoproteomics-based drug-discovery platform that enables multiplexed high-throughput screening of native proteins in cell extracts, thus preserving their post-translational modifications and protein interactions (Bergamini et al., 2012). Using affinity enrichment of target kinases afforded by immobilized ATP-competitive lipid kinase inhibitors, the potency of small-molecule test compounds was evaluated in competition binding assays. This approach allows targeting of proteins with low expression and measurements in human primary cells. The chemoproteomic strategy led to the design of CZC24832 [5-(2-amino-8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide], which exhibits superior selectivity for PI3Kγ than compounds reported previously (Camps et al., 2005; Bergamini et al., 2012). Despite this recent success, the lack of highly selective inhibitors of PI3Kγ has hampered detailed mechanistic studies of PI3Kγ in human primary cells and has hitherto precluded human clinical studies in inflammation, because adverse effects caused by the simultaneous inhibition of off-target kinases, in particular class IA PI3Ks, are intolerable for treatment of chronic non–life-threatening diseases.

C. Dual Phosphoinositide 3-Kinase γ/δ Inhibitors

Given the evidence outlined earlier from gene-targeted mice that PI3Kγ and PI3Kδ often work in concert and can have overlapping roles, dual inhibition of both targets with a single compound has been investigated. TargeGen (San Diego) described two diaminopteridine-diphenol-based compounds with good selectivity for both PI3Kγ and δ (Table 3). TG-101-110 [6- (1H-indol- 4-yl)-pteridin-2,4-diamine::6-(1H-indol-4-yl)pteridine-2,4-diamine] inhibits PI3Kγ and -δ but also displays PI3Kβ inhibition close to the IC₅₀ values for PI3Kγ and -δ (Doukas et al., 2006). TG-100-115 (3-[2,4-diamino-7-(3-hydroxyphenyl)pteridin-6-yl]phenol) has a better selectivity profile for PI3Kγ and -δ over PI3Kα and -β and shows minimal activity on a range of other protein kinases (Doukas et al., 2006). TG-100-115 was initially tested for safety and efficacy in a phase I/II clinical trial for patients with myocardial infarct. Although this compound does not seem to have been developed further, Infinity Pharmaceuticals (Cambridge, MA) and Intellikine have ongoing phase I trials with IPI-145 [N-(6-(4-amino-1-((8-methyl-1-oxo-2-o-tolyl-1,2-dihydroisoquinolin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[d]thiazol-2-yl)acetamide] (Tables 2 and 3). This compound represents the only PI3Kδ/-γ inhibitor currently in clinical development (Norman, 2011).

D. Phosphoinositide 3-Kinase β Inhibitors

TGX-221 [9-(1-anilinoethyl)-7-methyl-2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-one] is a PI3Kβ isoform-specific inhibitor developed initially as a novel therapeutic agent for the treatment of thrombosis (Jackson et al., 2005; Straub et al., 2008). The series of PI3Kβ inhibitors exemplified by TGX-221 was designed around substitutions of chemical groups of LY294002 that convey selectivity for PI3Kβ over other PI3K isoforms (Table 3) (Jackson et al., 2005). TGX-221 is effective in treating thrombus formation by platelets in vivo in both rats and mice. There are species differences in the effect of TGX-221 treatment on homeostatic bleeding times, which are unaffected in rats (Jackson et al., 2005) but increased in mice (Bird et al., 2011). PI3Kβ inhibition should therefore be approached with caution. However, the increasing recognition of a role for PI3Kβ in the immune system would suggest therapeutic applications beyond those initially envisaged for thrombosis. Evidence of cooperation between the β and δ isoforms suggests that development of dual PI3Kβ/δ inhibitors may have therapeutic promise; indeed, compounds with such selectivity have been reported, suggesting that this approach is feasible (Knight et al., 2006).

A. B-Cell Malignancies

PI3Kδ is a signaling hub for diverse stimuli in B lymphocytes, including the B-cell antigen receptor, the CD19 coreceptor, cytokines, chemokines, and Toll-like receptors (Fruman and Rommel, 2011). Mouse models of PI3Kδ deficiency indicated a key role in multiple aspects of B-lymphocyte biology and function (Table 1), including development, proliferation, and cell survival (Fruman and Rommel, 2011). These models also indicated that acute inhibition of PI3Kδ could be prove efficacious against various pathological B-cell conditions, including lymphoma. CAL-101 has been studied extensively in patients with relapsed or refractory B-cell malignancies. There are at least 15 types of mature B-cell lymphoma that can have distinct pathological and clinical features and hence may often require diverse treatment strategies (Küppers, 2005). In preclinical studies using chronic lymphocytic leukemia (CLL) cell lines and primary patient CLL samples, CAL-101 blocked constitutive PI3K signaling resulting in decreased phosphorylation of Akt and decreased cell viability of cell lines. In patient samples of CLL, there is increased PI3Kδ expression and activity that is reduced by CAL-101. These effects have been observed across a broad range of other immature and mature B cell malignancies, including CD5⁺ mantle zone B-cell lymphomas, follicular lymphomas and multiple myeloma (Herman et al., 2010; Ikeda et al., 2010; Fruman and Rommel, 2011; Hoellenriegel et al., 2011; Lannutti et al., 2011). Ongoing clinical evaluation of CAL-101 has revealed that it causes rapid lymph node shrinkage and lymphocytosis (Fruman and Rommel, 2011; Hoellenriegel et al., 2011; Lannutti et al., 2011). This inhibitor has therefore demonstrated an essential role for PI3Kδ in constitutive PI3K signaling that is required for the survival of several different types of malignant B cells. Oncogenic mutations of components of the PI3K signaling pathway are infrequent in B-cell malignancies. A potential mechanism for PI3K activation in this setting is tonic antigen-independent B-cell receptor (BCR) signaling that requires PI3Kδ for the transduction of proliferation and survival signals.

The malignant B-cell microenvironment is important for disease progression and CAL-101 was tested in assays that model CLL microenvironment interactions in vitro. CAL-101 inhibited CLL cell chemotaxis toward CXCL12 and CXCL13 in Boyden chamber type assays and migration beneath stromal cells (pseudoemperipolesis) (Hoellenriegel et al., 2011). This is consistent with data from mouse models indicating that PI3Kδ is the dominant isoform required for B-cell migration (Reif et al., 2004). CAL-101 also down-regulated secretion of chemokines in stromal cocultures and after BCR triggering. CAL-101 reduced survival signals derived from the BCR or from nurse-like cells and inhibited BCR- and chemokine receptor-induced Akt and extracellular signal-regulated kinase activation in CLL cells. In stromal cocultures, CAL-101 sensitized CLL cells toward chemotherapies such as bendamustine (Hoellenriegel et al., 2011). These results are consistent with clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels and a surge in lymphocytosis during CAL-101 treatment. Hence, CAL-101 displays a dual mechanism of action in which it both decreases cell survival and reduces interactions that retain CLL cells in protective tissue microenvironments.

Hodgkin lymphoma (HL) is a malignant lymphoma of B-cell origin (Thomas et al., 2004). The malignant cells, known as Reed-Sternberg (RS) cells, represent less than 2% of the tumor mass, the remainder being composed of a mix of reactive inflammatory cells attracted by the RS cells. HL cell lines and primary samples from patients with HL have been reported to express high levels of PI3Kδ and constitutive PI3K pathway activation (Meadows et al., 2012). As with CLL, CAL-101 was able to reduce the positive interaction between stromal cells and malignant RS cells; this may be due, in part, to reduced release of the chemokine CCL5 by RS cells. Cell-cycle arrest and apoptosis was also induced in HL cell lines, and dual inhibition of PI3Kδ and mTOR may be of benefit in the treatment of HL.

B. Rheumatoid Arthritis

RA is a chronic autoimmune disease that predominantly affects joints of the hands and feet, causing immobilization and disability. In RA, the structure of the synovium is transformed into a pannus-like tissue that invades cartilage. The inflamed synovium consists of inflammatory cells, such as macrophages, neutrophils, and T and B cells, as well as hyperplasia of synovial lining cells, which are predominantly fibroblast-like synoviocytes. Strong granulocyte and lymphocyte recruitment into these joints is one of the major causes of the onset of RA (Firestein, 2003). Damage to the synovial tissue is driven by aggressive inflammatory cytokine signaling in these joints. Given that PI3Kγ has a pivotal role in mediating leukocyte migration (Hirsch, 2000; Sasaki et al., 2000a; Reif et al., 2004; Smith et al., 2007; Lim et al., 2009; Thomas et al., 2009) and activation as well as mast-cell degranulation (Ali et al., 2008; Willox et al., 2010), it was predicted that blocking PI3Kγ might be an effective strategy to fight RA. This was explored with both genetic and pharmacological approaches (using the PI3Kγ-selective inhibitor AS-605240) in distinct animal models of arthritis. When arthritis was induced by injection of monoclonal antibodies to type II collagen (a model that focuses on the effector phase of arthritis), PI3Kγ-null mice exhibited reduced paw swelling, synovial inflammation, and cartilage erosion. In wild-type mice, oral administration of AS-605240 effectively inhibited antibody-induced inflammation and cartilage destruction to a degree similar to that observed in the PI3Kγ-null mice (Camps et al., 2005). When arthritis was induced by injection of bovine type II collagen along with adjuvant, (a model in which an adaptive immune response contributes to disease development similarly to the human disease), AS-605240 reduced symptoms correlating with decreased neutrophil infiltration (Camps et al., 2005). CZC24832 also shows anti-inflammatory effects in a collagen-induced arthritis model that correlated with reduced Th17 differentiation, a pro-inflammatory helper T-cell type characterized by expression of the cytokine IL-17 (Weaver and Murphy, 2007). Indeed, CZC24832 treatment also led to reduced IL-17 production (Bergamini et al., 2012). This confirms the long-held belief that pharmacological inactivation of PI3Kγ alone can lead to amelioration of inflammatory disease.

Although widely used as a disease model for RA, murine collagen-induced arthritis in mice reflects mainly the immunological components of the disease and constitutes an acute T-cell-mediated autoimmune arthritis. Therefore, alternative animal models of RA have been developed that resemble the chronic, destructive phase of RA. For example, transgenic overexpression of human tumor necrosis factor α leads to a chronic inflammatory destructive polyarthritis that is similar to human RA and is sensitive to blockade of TNFα by treatment with neutralizing anti-TNFα antibodies. Loss of PI3Kγ in human TNFα transgenic mice led to reduced arthritis compared with control mice (Hayer et al., 2009). It is noteworthy that PI3Kγ deficiency does not alter the recruitment of inflammatory cells as observed in the collagen-induced arthritis model of RA, but it significantly reduces cartilage damage through reduced expression of matrix metalloproteinases in fibroblasts and chondrocytes (Hayer et al., 2009). In vitro analyses demonstrate that the decreased invasiveness of fibroblasts is mediated by reduced phosphorylation of Akt and extracellular signal-regulated kinase. PI3Kγ expression is significantly higher in the synovia of patients with RA compared with that from patients with osteoarthritis. Furthermore, inhibition of PI3Kγ using AS-252424 (5-[[5-(4-fluoro-2-hydroxyphenyl)-2-furanyl]methylene]-2,4-thiazolidinedione) reduced TNFα-induced matrix metalloproteinase production in fibroblasts isolated from patients with RA (Hayer et al., 2009). This study, therefore, provides further mechanistic insights into PI3Kγ in RA that extends beyond its established role in immune cell migration.

Recent studies have revealed that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis synovium, and PI3Kδ mRNA can be selectively induced in cultured synoviocytes by inflammatory cytokines. Indeed, inhibition of PI3Kδ diminished PDGF-mediated synoviocyte growth (Bartok et al., 2012). In a K/BxN-serum transfer model of arthritis, in which neutrophils and LTB4 participate in the effector phase of the inflammatory arthritis, genetic deletion, or selective inhibition of PI3Kδ diminishes joint erosion to a level comparable with its PI3Kγ counterpart. Induction and progression of joint destruction was profoundly reduced in the absence of both PI3K isoforms and is consistent with both isoforms being required for LTB4-mediated neutrophil chemotaxis, as described previously (Randis et al., 2008).

Important differences between PI3Kγ and PI3Kδ have been noted in the mechanisms underpinning joint destruction. For example, in a model of osteoclastogenesis, the PI3Kδ-selective inhibitor IC-87114 significantly inhibited the generation of osteoclasts, whereas selective inhibition of PI3Kγ with AS-605240 had no effect (Toyama et al., 2010). Taken together, these lines of evidence suggest that dual inhibition of PI3Kγ and PI3Kδ would be more therapeutically beneficial than targeting one isoform alone. However, in the same K/BxN model, PI3Kβ-null mice were also partially protected, whereas mice that were also deficient in PI3Kδ activity were highly resistant to disease (Kulkarni et al., 2011). Hence, inhibition of PI3Kβ (alone or in combination with targeting of PI3Kδ) may also offer potential in the treatment of RA in certain situations.

C. Systemic Lupus Erythematosus

SLE is a chronic inflammatory disease, characterized at early stages by an increase in long-lasting, autoreactive memory CD4⁺ T lymphocytes (Kuroiwa and Lee, 1998; Wakeland et al., 1999). Dysregulated T cells lead to polyclonal B cell activation, generalized B-cell expansion, hypergammaglobulinemia, and increased autoantibody production. Circulating anti-DNA antibodies form complexes that are retained in kidneys and locally activate the complement cascade. As the disease progresses, T cells and macrophages infiltrate the kidney and amplify the local inflammatory response culminating eventually in glomerulonephritis and renal failure (Kuroiwa and Lee, 1998; Wakeland et al., 1999).

Mouse SLE involves abnormal activation of CD4⁺ T cells that accumulate as activated memory cells and contribute to disease pathogenesis (Jevnikar et al., 1994; Borlado et al., 2000; Lang et al., 2003). Deletion of PI3Kγ in this model reduced the survival of pathogenic memory CD4+ T lymphocytes, which ultimately led to a reduction in disease (Barber et al., 2005). In the MRL^lrp/lpr multigenic SLE-prone mouse model (in which SLE susceptibility correlates with mutations at several loci, as for human SLE5), AS-605240 and related compounds increased survival and reduced autoantibody production, proteinuria, and glomerulonephritis. Mice treated with AS-605240 showed no adverse side effects after 3 months of treatment (Barber et al., 2006). Mice expressing an activating mutation of the p85 regulatory subunit p65(PI3K) in T cells develop an SLE-like disease because of the increased survival of mature CD4⁺ T lymphocytes (Borlado et al., 2000). Deletion of PI3Kγ in this model reduced the survival of pathogenic memory CD4⁺ T lymphocytes. Together, these results validate the PI3Kγ isoform as a target for SLE treatment (Barber et al., 2005, 2006). More recently however, increased PI3Kδ but not -γ activity was observed in patients with SLE. This increased activity made activated and memory T cells more resistant to activation induced cell death. This resulted in an increased number of memory T cells and highlights an important role for PI3Kδ in SLE (Suárez-Fueyo et al., 2011).

D. Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is an induced method of autoimmune inflammation of the central nervous system (CNS) commonly used to model diseases such as MS in rodents (Seil, 1972; Finsen et al., 2002). EAE is characterized by infiltration of inflammatory cells into the CNS that drive destruction of neurons, causing MS-like symptoms, including ataxia, paralysis of limbs, and loss of weight.

Until recently, MS and EAE were thought to be driven by autoreactive Th1 cells. However, considerable evidence now indicates that the T-cell lineage most likely to be driving EAE pathogenesis are Th17 cells (Kleinschek et al., 2007). In a Th17-driven EAE model, the absence of PI3Kγ delayed progression of motor dysfunction (Rodrigues et al., 2010). Lower levels of the proinflammatory chemokines CCL2 and CCL5 were observed in the meninges of PI3Kγ-deficient mice after EAE induction. Consequently, there were markedly reduced numbers of infiltrating immune cells. Both genetic and pharmacological targeting of PI3Kγ indicated that it plays a more important role in mediating leukocyte survival than in mediating leukocyte adhesion in this experimental model of MS. However, using the same EAE model, another group (Haylock-Jacobs et al. 2011) reported that signaling through PI3Kδ is required for full and sustained pathologic features of EAE. In PI3Kδ-inactivated mice, T-cell activation and function during EAE was markedly reduced, and fewer T cells were observed in the CNS. Reminiscent of observations made in the PI3Kγ deficient mice, there were significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity. The PI3Kδ inhibitor IC-87114 also had greater inhibitory effects on Th17-cell generation in vitro than on Th1-cell generation. Taken together, these data indicate that both PI3Kγ and PI3Kδ can contribute to the pathogenesis of EAE, influencing cell survival, differentiation, and migration mechanisms (Haylock-Jacobs et al., 2011). Thus, dual targeting of PI3Kγ and -δ might be a therapeutic option for the treatment of EAE.

E. Airway Disorders

Asthma is a chronic disorder in which the airway occasionally constricts, becomes inflamed, and is lined with excessive amounts of mucus, often in response to one or more triggers, such as exposure to an allergen. Airway eosinophilia, mucus accumulation, elevated serum IgE levels, and airway hyper-responsiveness are fundamental characteristics of allergic asthma. Th2 cells, together with other inflammatory cells such as mast cells, neutrophils, B cells, and eosinophils, play an essential role in the pathophysiology of this disease (Medina-Tato et al., 2006, 2007). With regard to asthma, inhibition of PI3Kδ may be of primary importance. For example, the early-phase response in asthma is largely driven by mast-cell degranulation (Bloemen et al., 2007), and although both PI3Kδ and -γ contribute to mast-cell activation (Laffargue et al., 2002; Ali et al., 2008), the former seems to predominate (Ali et al., 2008). Subsequent events during the late-phase response, such as IgE hyperproduction, may also depend primarily on PI3Kδ (Okkenhaug et al., 2002; Al-Alwan et al., 2007). In contrast, lipopolysaccharide (LPS) and smoke-induced pulmonary inflammations are probably driven by chemokine-induced neutrophil recruitment, and PI3Kγ is the isoform primarily responsible for this response (Thomas et al., 2005).

In an ovalbumin (OVA) model of asthma, PI3Kδ^D910A mice or treatment with the PI3Kδ inhibitor IC-87114 leads to decreased Th2 cytokine and mucus production as well as reduced eosinophil recruitment, airway remodeling, and hyper-responsiveness (Lee et al., 2006; Nashed et al., 2007; Farghaly et al., 2008). More recently, it has become apparent that pharmacological inhibition of PI3Kδ with IC-87114 also reduces the levels of IL-17 in the OVA-induced mouse model (Park et al., 2010). GlaxoSmithKline has developed an inhaled PI3Kδ inhibitor that, in the OVA model, reduced IL-13 levels and eosinophilia in bronchoalveolar lavage fluid, and effects were comparable with treatment with corticosteroids. It is noteworthy that in an in vitro steroid-insensitive assay of asthma, this PI3Kδ inhibitor was still active (Amour et al., 2011). In a model of allergic airway inflammation induced by cockroach antigen, IC-87114 significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyper-responsiveness, reduced mucus secretion, and expression of various pro-inflammatory molecules (Kang et al., 2012). In vitro observations, suggested that the reduced eosinophil recruitment observed in IC-87114-treated cockroach antigen-challenged mice, may be caused by a more direct effect of the inhibitor on eosinophil trafficking (rolling, adhesion, and migration) rather than Th2 cytokines and eosinophil-active chemokines (Kang et al., 2012). This is in contrast to observations in the OVA model, in which IC-87114 treatment resulted in a significant reduction in Th2 cytokines (Lee et al., 2006).

There is also strong evidence that PI3Kγ contributes to the immune processes that underpin allergen-induced airway inflammation. In the OVA-induced model of asthma, challenge of PI3Kγ-null mice with allergen resulted in a greatly reduced number of infiltrating leukocytes in their bronchoalveolar lavage fluid compared with wild type, as well as decreased hyper-responsiveness, airway remodeling, and fibrosis (Lim et al., 2009; Takeda et al., 2009; Thomas et al., 2009). In addition, PI3Kγ seems crucial for eosinophil-mediated inflammation in a mouse model of pleurisy involving intrapleural administration of OVA (Pinho et al., 2005). Given evidence implying a role for both PI3Kγ and -δ in allergic airway inflammation in mice, it is interesting to note that the dual PI3Kγ/δ inhibitor TG-100-115 reduced airway inflammation in the OVA model (Doukas et al., 2009).

Other airway disorders may also benefit from the targeting of PI3Kγ and -δ. For example, PI3Kδ-selective inhibitor CAL-101 has been tested in a clinical trial for allergic rhinitis (ClinicalTrials.gov identifier NCT00836914). Furthermore, other non–allergen-based mouse models of airway inflammation exploiting knockout mice have revealed that PI3Kγ is required for neutrophil recruitment in both chemokine airway instillation and intraperitoneal LPS models of lung injury in PI3Kγ-null mice (Yum et al., 2001; Thomas et al., 2005). Likewise, the dual PI3Kγ/δ inhibitor TG-100-115 reduced neutrophilia and TNFα production in response to LPS and was effective as an intervention after cigarette smoke exposure (Doukas et al., 2009). However, it remains unclear whether the intrapulmonary TG100-115 levels reported in this study led to inhibition of PI3K isoforms other than PI3Kγ, thereby making the task of assigning biological outcomes to inhibition of specific isoforms difficult. This work supports the value of a dual-specificity inhibitor such as TG100-115 in treating distinct respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). The smoke model is of particular interest because, like human COPD, it is steroid-resistant. Steroid resistance is the result of several alterations within the cell, including dysregulation of the glucocorticoid receptor, attenuated histone deacetylase activity, and increased proinflammatory gene transcription (Adcock et al., 2008). The demonstration that aerosolized TG100-115 provides efficacy as an interventional therapy in a smoke-induced neutrophilia model therefore suggests the potential use of TG100-115, not only for patients with COPD but also for steroid-resistant patients with severe asthma (Adcock et al., 2008).

Despite the encouraging preclinical and clinical data relating to use of inhibitors targeting the δ and γ isoforms of PI3K, there are some potential caveats. For example, PI3Kδ negatively regulates B cell antibody class switching (Zhang et al., 2008). One potential cause for concern is that increased IgE levels were recorded when PI3Kδ is inactivated or inhibited with IC-87114, because IgE drives hyper-responsiveness. In contrast, loss of PI3Kγ did not have this effect (Takeda et al., 2009).

PI3K has also been proposed as a respiratory disease target on the basis of its role in mediating smooth muscle and endothelial cell proliferation and, by extension, airway remodeling (Goncharova et al., 2002; Krymskaya, 2007); this represents another area worthy of future research. Indeed, a role for the α and β isoforms has recently been proposed in remodeling of airway smooth muscle cells, a key process involved in asthma. PIK75 (N-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide hydrochloride), a PI3Kα-selective inhibitor, demonstrated a role for this isoform in fibronectin deposition and survival of asthmatic cells in response to TGFβ treatment. In addition, PI3Kα and, in part, PI3Kβ also facilitated secretion of VEGF and IL-6 from these cells (Moir et al., 2011).

F. Myocardial Infarction

When blood flow to the heart is reduced as a result of cardiovascular disease, this causes myocardial ischemia, which can lead to apoptosis of cardiac myocytes. A reperfusion event restores blood flow, but oxidative damage results in an increase in inflammatory immune cell infiltration and tissue damage. This wave of ischemia/reperfusion events eventually leads to myocardial infarction (MI) (Frangogiannis et al., 2002). Given evidence for inflammatory activation as an important pathway in disease progression in chronic heart failure, the PI3Kγ and PI3Kδ isoforms have been considered potential therapeutic targets. Indeed, in both rat and porcine occlusion models of MI, dual inhibition of PI3Kγ and -δ using TG-100-115 showed promising therapeutic benefit (Doukas et al., 2006). Inhibition of both isoforms causes a decrease in inflammatory cell infiltration in vivo and a reduction in edema and disease severity.

Atherosclerosis is a chronic disease of large arteries and is the primary cause of MI and stroke (Hansson and Libby, 2006). PI3Kγ levels are reported to be increased in atherosclerotic lesions, and a lack of PI3Kγ was found to protect against atherosclerosis (Fougerat et al., 2008). This was due in part to a reduced ability of PI3Kγ-null T cells and macrophages to infiltrate into lesions. In addition, loss of PI3Kγ also caused the lesions to have an increase in collagen and smooth muscle. This resulted in more stable plaques in PI3Kγ knockout mice, which were less likely to form a thrombus (Fougerat et al., 2008). PI3K, along with Erk1/2, integrates monocyte chemotactic protein-3 signaling to promote smooth-muscle cell proliferation, a process important for thickening of artery walls in atherosclerosis (Maddaluno et al., 2011). Accordingly, the PI3Kγ-selective inhibitor AS-605240 was effective in treating mouse models of established atherosclerosis (Chang et al., 2007; Fougerat et al., 2008). PI3K also has important roles in macrophage function in atherosclerotic legions. Use of AS-605240 or PI3Kγ-null mice demonstrated that PI3Kγ is partially required for low-density lipoprotein uptake and cholesterol accumulation in macrophage foam cells (Anzinger et al., 2012). However, PI3K signaling is also required for the action of liver X receptor ligands, which cause the efflux of cholesterol from foam cells and prevent development of atherosclerotic legions (Huwait et al., 2011).

A. Class II Phosphoinositide 3-Kinases

Class II PI3Ks (comprising the α, β, and γ isoforms) are structurally distinct members of the PI3K family that have received relatively little attention compared with their class I cousins (Fig. 1). The primary product of class II PI3Ks is generally considered to be phosphatidylinositol 3-phosphate [PI(3)P], although this is largely based on in vitro evidence (Falasca and Maffucci, 2012). Although PI3KC2α and PI3KC2β have a wide tissue distribution and are both expressed in leukocytes, PI3KC2γ has a more restricted pattern of expression and is absent from most leukocytes.

Unlike the class I PI3Ks, the class II PI3Ks are activated by agonist-induced relocalization of constitutively active class II enzyme to the plasma membrane via interaction of their N and C termini with adaptor signaling molecules, such as Grb2 in epidermal growth factor receptor signaling (Wheeler and Domin, 2001; Mazza and Maffucci, 2011). Agonists of other receptor tyrosine kinases and GPCRs as well as membrane-associated complexes have also been described as activators of class II PI3Ks as shown in Fig. 5 (Turner et al., 1998; Gaidarov et al., 2001; Arcaro et al., 2000, 2002; Maffucci et al., 2005; Das et al., 2007).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

Activation and function of class II and III PI3Ks. PI(3)P is a product of both class II and III PI3K enzymes, although the structure and the mechanisms of activation of these enzymes are different. Stimulation of cells with agonists for receptor tyrosine kinases including EGF, stem cell factor, and insulin or activation of chemokine GPCRs causes relocation of constitutively active class II PI3Ks to the plasma membrane (Wheeler and Domin, 2001; Mazza and Maffucci, 2011). Membrane-associated complexes, such as clathrin, intersectin (not depicted), and FROUNT are also involved in class II relocalization (Gaidarov et al., 2001; Terashima et al., 2005; Das et al., 2007; Toda et al., 2009). Nutrients such as amino acids can activate the class III enzyme Vps34, which, through PLD, promotes mTORC1 signaling (Byfield et al., 2005; Nobukuni et al., 2005; Yoon et al., 2011). Vps34 can also be tyrosine-phosphorylated in a Src-dependent manner. Little is known about the function of both enzyme families; however, the class II PI3KC2β has roles in migratory process downstream of CCR2 and CCR5 chemokine GPCRs (Domin et al., 2005; Maffucci et al., 2005; Cai et al., 2011) as well as the positive regulation of KCa3.1 downstream of TCR activation. The latter is critical for maintenance of negative membrane potential that facilitates sustained calcium entry into T cells via calcium release-activated Ca²⁺ channels. Increased cytosolic Ca²⁺ then mediates the transcriptional activation of a number of genes critical for T-cell activation. PI(3)P generated by Vps34 is required for vesicle and IL-7RA trafficking, autophagy, as well as cell transformation and growth (McLeod et al., 2011). Although selective class II inhibitors have yet to be described, the dual class I/mTOR inhibitor PI-103 has been shown to inhibit PI3KC2β. Inhibitors that display limited (3MA) or more selectivity (PIK-93) for Vps34 have also been described (Miller et al., 2010).

One of the hallmark features of class II PI3Ks is their relatively low sensitivity to the classic PI3K inhibitors wortmannin and LY294002 (Domin et al., 1997). Consequently, the involvement of class II PI3Ks in biological processes may well have been underestimated. Conversely, some class I PI3K inhibitors, such as the PI3Kα/mTOR inhibitor PI-103 [3-(4-morpholin-4-ylpyrido[2,3]furo[2,4-b]pyrimidin-2-yl)phenol], actually exhibit comparable activity toward PI3KC2β but, crucially, are much less potent toward PI3KC2α (Knight et al., 2006). Progress in understanding the biological role of class II PI3Ks has been relatively slow compared with that for the four class I isoforms. This is due in part to the paucity of class II PI3K-selective pharmacological tools and the lack of a knockout mouse. However, the recent report of a PI3KC2α knockout mouse will hopefully shed light on the biological role of this enzyme (Harris et al., 2011a).

Despite the lack of selective pharmacological tools with which to target class II PI3Ks, molecular approaches have implicated the class II PI3KC2β and its primary product PI(3)P in the regulation of cell adhesion, actin reorganization, and migration in nonimmune cell systems (Domin et al., 2005; Maffucci et al., 2005). Certainly, class II PI3K isoforms are activated by CCR2 chemokine agonists in monocytic cell lines (Turner et al., 1998). The mechanism of interaction of class II PI3K with GPCRs is unclear, but the protein FROUNT, which is structurally similar to clathrin and facilitates leukocyte infiltration in response to CCR2 and CCR5, may be one possible mediator (Gaidarov et al., 2001; Terashima et al., 2005; Toda et al., 2009). Class II PI3KC2β, but not PI3KC2α, has been demonstrated to play an important and unexpected role in CD4⁺ T-cell activation downstream of the TCR (Srivastava et al., 2009; Cai et al., 2011).

B. Class III Phosphoinositide 3-Kinases

The single class III PI3K Vps34 exhibits considerable homology with the catalytic subunits of other PI3Ks, particularly at the level of domain organization. Vps34 has a structurally uncharacterized N-terminal region of approximately 50 amino acids, followed by C2, helical, and kinase domains that are approximately 30% homologous with PI3Kγ, but it has no Ras binding domain (Vanhaesebroeck et al., 2010). Vps34 enzymes are unique among PI3Ks in that they will only use phosphatidylinositol as a substrate. Vps34 could therefore share protein effectors with the class II PI3Ks, but it is not clear whether the functions of class II and class III PI3Ks overlap.

Vps34 is thought to be a potential anticancer target as a result of its roles in autophagy (Burda et al., 2002; Backer, 2008; Simonsen and Tooze, 2009) and its involvement in regulating nutrient input into the mTORC/S6 kinase 1 signaling pathway (Byfield et al., 2005; Nobukuni et al., 2005; Yoon et al., 2011). Furthermore, Vps34 is tyrosine-phosphorylated by Src, and this phosphorylation is essential for Src-mediated cellular transformation and anchorage-dependent growth. Protein levels of Vps34 also correlate with tumorigenic activity of human breast cancer cells (Hirsch et al., 2010). Information relating to the role of Vps34 specifically in immune cells is limited, but class III (along with class II) PI3Ks regulate various aspects of vesicle trafficking, so there is obvious potential for their involvement in activities such as antigen processing and cytotoxic responses. Indeed, Vps34 has recently been shown to be required for the trafficking of IL-7Rα to the surface of naive T lymphocytes, a process key for their survival (McLeod et al., 2011). Vps34's role in autophagy suggests it may prove important for immune recognition of tumor antigens, regulation of T-cell homeostasis, and immune tolerance (Li et al., 2008; Nedjic et al., 2008; Walsh and Edinger, 2010). There is considerable evidence that class III PI3K is important for phagocytosis because PI(3)P accumulates on phagosomal membranes (Vieira et al., 2001) and contributes to phagosomal maturation and pathogen destruction (Fratti et al., 2001; Vieira et al., 2001; Ellson et al., 2006b). Indeed, some virulent strains of bacteria (e.g., Mycobacterium tuberculosis) are thought to evade host defenses by interfering with PI(3)P metabolism (Deretic et al., 2006). The binding of PI(3)P to the PX domain of p40phox plays an important role in phagosomal oxidase activation during engulfment of serum-opsonized S. aureus and IgG-opsonized particles (Ellson et al., 2006a; Ellson et al., 2006b; Suh et al., 2006). Although the source of phagosomal PI(3)P accumulation may not necessarily be restricted to Vps34, small interfering RNA targeting of Vps34 has revealed that this enzyme is responsible for the synthesis of PI(3)P on phagosomes containing S. aureus and Escherichia coli (Anderson et al., 2008). Thus, there may be opportunities to target Vps34 in destructive inflammatory/autoimmune diseases in which there is dysregulated phagosomal activity and antigen presentation of self-molecules, for example.

As with the class II PI3Ks, the lack of Vps34-specific inhibitors has restricted our understanding of the biological/pathogenic importance of Vps34. It is noteworthy that both yeast and human Vps34 share a lysine residue that, in human class I PI3Ks, is the site of covalent modification by the pan-PI3K inhibitor wortmannin (Wymann et al., 1996; Walker et al., 2000). Indeed, Vps34 is inhibited by both wortmannin and LY294002 (Backer, 2008), and although 3-methyladenine (3MA) has been suggested to be a specific inhibitor of hVps34, in fact it inhibits class I and II PI3Ks as well (Ito et al., 2007). However, the crystal structure of Vps34 in complex with 3MA has been solved (Miller et al. 2010). 3MA is often used as an inhibitor of autophagy, and the IC₅₀ values for Vps34 and PI3Kγ are 25 and 60 mM, respectively (Miller et al., 2010). 3MA has a slight preference for the hydrophobic ring comprising Phe673, Tyr746, and Leu812 that encircles the 3-methyl group of 3MA and that is unique and specific to Vps34. Hence, 3MA may be a useful lead for development of compounds with improved selectivity for Vps34 over other PI3K isoforms (Miller et al., 2010).

A. Role of SH2-Domain Containing Inositol-5-phosphatase-1 in the Immune System

SHIP-1 translocates to the plasma membrane after surface receptor stimulation and hydrolyzes the PI3K-generated second messenger PI(3,4,5)P₃ to PI(3,4)P₂. As a result, SHIP-1 is able to modulate PI(3,4,5)P₃-mediated signaling and hence the proliferation, differentiation, survival, activation, and migration of hematopoietic cells (Harris et al., 2008). SHIP-1 has been implicated in signaling pathways triggered by cytokine, chemokine, antigen, and IgG engagement in a variety of immune cells (Lioubin et al., 1994; Liu et al., 1994; Harris et al., 2008). Genetic analysis of SHIP-1 mutant mice (Table 1), has revealed a pivotal role for SHIP-1 in regulating the receptor repertoire and cytolytic function of NK cells, B lymphocyte development and antibody production, the myeloid cell response to bacterial mitogens, development of marginal zone macrophages, lymph node recruitment of dendritic cells, and mast cell degranulation (Leung et al., 2009). SHIP-1 also plays a critical role in homeostasis of myeloid and lymphoid effector and regulatory cells (Ghansah et al., 2004; Rauh et al., 2005; Locke et al., 2009; Kuroda et al., 2011) and plays an important role in establishing endotoxin tolerance in macrophages (Sly et al., 2004) as well as regulating leukocyte polarization during migratory responses (Harris et al., 2011b).

The key regulatory role of SHIP-1 has been exploited by several opportunistic pathogens that target these phosphatases to evade immune detection. Thus, lymphocytes are particularly sensitive to the cytolethal distending toxin subunit B (CdtB), an immunotoxin produced by Actinobacillus actinomycetemcomitans, that can hydrolyze PI(3,4,5)P₃ to PI(3,4)P₂. Exposure to CdtB leads to cell-cycle arrest and death by apoptosis. The lipid phosphatase activity of CdtB may therefore result in reduced immune function, facilitating chronic infection with A. actinomycetemcomitans and other enteropathogens that express Cdt proteins (Shenker et al., 2007). The measles virus evades destruction by the immune system, at least in part, by targeting negative regulation of PI3K/Akt signaling. It induces expression of the SHIP-1 homolog SIP-110, which depletes cellular PI(3,4,5)P₃ pools, suggesting that the threshold for activation signals leading to induction of T-cell proliferation is raised (Avota et al., 2006). The predominant expression in hematopoietic cells coupled with targeting of this protein by pathogens to avoid immune recognition suggests that SHIP-1 might offer opportunities for the design of new drugs targeting PI3K-dependent signaling.

B. SH2-Domain Containing Inositol-5-phosphatase-1 Can Act as a Tumor Suppressor in Hematological Malignancies and Is Crucial to Antitumor Immune Responses

The PI3K-dependent signaling pathway has a well established role in regulating cell survival, proliferation, and differentiation (Manning and Cantley, 2007). As we have seen, the PI3K/Akt/mTOR pathway is one of the most commonly activated pathways in human cancer (Engelman et al., 2006; Manning and Cantley, 2007). Indeed, there has been a long appreciation that one of the negative regulators of PI3K signaling, the 3′ lipid phosphatase PTEN, is frequently lost in many cancers (Hollander et al., 2011). Likewise, SHIP-1 expression is frequently lost, down-regulated, or mutated in many hematological malignancies, including acute myeloid leukemia (Luo et al., 2003). Scanning of the SHIP-1 3′ untranslated region has revealed perfect sequence complementarity with the seed sequence of miR-155. Elevated levels of miR-155 and consequent diminished SHIP-1 expression have been linked to B-cell lymphomas (Rodriguez et al., 2007; Costinean et al., 2009; O'Connell et al., 2009). In addition, it has been reported that oncogenic proteins, including BCR/Abl (implicated in chronic myelogenous leukemia), induce SHIP-1 down-regulation by a variety of mechanisms (Ruschmann et al., 2010). Consistent with its role as a tumor suppressor, SHIP-1 restricts development of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Ghansah et al., 2004; Locke et al., 2009). Thus, SHIP-1 deficiency leads to an expansion of MDSCs and regulatory T cells and, hence, suppression of antitumor immune responses. This may be another mechanism for increased tumorigenesis if SHIP-1 expression is reduced. However, the role of SHIP-1 in leukemia seems more complex than initially thought. In this regard, there is evidence that SHIP-1 can actually support cancer cell survival as a small-molecule inhibitor of SHIP-1 induces apoptosis of multiple myeloma cells (Brooks et al., 2010). This is consistent with its production of PI(3,4)P₂, which is known to facilitate Akt activation and thereby cell proliferation, survival, and tumorigenesis (Manning and Cantley, 2007). Others have shown that SHIP-1 inhibits CD95/APO-1/Fas-induced apoptosis in T cells by promoting CD95 glycosylation independently of its phosphatase activity (Charlier et al., 2010).

A. Allosteric SH2-Domain Containing Inositol-5-phosphatase-1 Activators

SHIP-1 is a particularly ideal target for development of potential therapeutics for treating immune and hematopoietic disorders because its hematopoietic-restricted expression would limit the effects of a specific SHIP-1 agonist to target cells, and hence would probably minimize off-target tissue effect. One would predict, for example, that activators of SHIP-1 would lead to a reduction of cellular PI(3,4,5)P₃ and hence mimic the effect of PI3K inhibitors. In 2005, the natural product pelorol [methyl(4aS,6aR,11aR,11bS)-9,10-dihydroxy-4,4,6a,11b-tetramethyl-1,2,3,4a,5, 6,11,11a-octahydrobenzo[a]fluorene-7-carboxylate] extracted from the tropical marine sponge Dactylospongia elegan, was identified as the first SHIP-1 activator (Yang et al. 2005). Pelorol and subsequent synthetic derivatives were found to bind allosterically to a newly identified C2 domain of SHIP-1, leading to enzyme activation. Two of these compounds based on the structure of pelorol, AQX-016A [(6aR,11aR,11bS)-4,4,6a,7,11b-pentamethyl-1,2,3,4a,5, 6,11,11a-octahydrobenzo[a]fluorene-9,10-diol] and AQX-MN100 were effective in the inhibition of immune cell activation and reducing inflammation in vivo using a model of endotoxin shock in mice (Ong et al., 2007). Moreover, these molecules have been successfully used to kill multiple myeloma cells in vitro indicating that SHIP-1 agonists could be effective anticancer agents (Kennah et al., 2009).

Aquinox Pharmaceutical Inc. (Richmond, BC, Canada) is currently developing a small-molecule SHIP-1 activator to exert negative effects on the PI3K pathway and that is intended for clinical use. A lead compound termed AQX-1125 with an undisclosed structure, has been shown to reduce Akt phosphorylation in the leukemic T cell line MOLT-4 and murine splenic B cells (Stenton et al., 2011). As expected, AQX-1125 had no effect on Jurkat cells, another leukemic T cell line that has previously been demonstrated to be deficient in SHIP-1 expression (Astoul et al., 2001). AQX-1125 was able to inhibit the chemotactic response of numerous human leukocyte populations, including activated T cells and neutrophils. It is noteworthy that AQX-1125 exhibited potent anti-inflammatory effects, significantly reducing OVA-induced leukocyte lung infiltration in Brown Norway rats (Stenton et al., 2011). Hence, these results indicate promising potential for SHIP-1 activators in the treatment of inflammatory disorders. AQX-1125 has passed phase I clinical trials in 2011, with phase IIa clinical studies initiated in late 2011 for the treatment of mild and moderate asthma (Aquinox Pharmaceuticals, 2011). With regard to the latter, the recent finding that TLR stimulation augments IgE plus Ag-induced TNFα and IL-6 production from mucosal mast cells (Ruschmann et al., 2012) might explain the exacerbation of IgE-mediated allergic episodes by infectious agents (Qiao et al., 2006). Because IgE synergizes with TLR ligands to trigger cytokine production from SHIP-1-null mucosal mast cells, activating SHIP-1 specifically in these cells might be useful for treating chronic inflammatory diseases such as asthma.

B. SH2-Domain Containing Inositol-5-phosphatase-1 Inhibitors

A small-molecule inhibitor of SHIP-1 has also been reported, although its site of action is unclear (Brooks et al., 2010). Using high-throughput screening, 3α-aminocholestane (3AC) was identified as a potent SHIP-1 inhibitor. Treatment of mice with 3AC led to increased numbers of MDSCs that repress allogeneic T-cell responses. Indeed, 3AC reduced ability of peripheral lymphoid tissues to prime myeloid-associated responses and protected against graft-versus-host disease, which involves priming of allogeneic T cells and is a common complication arising after bone marrow transplantation. The results with 3AC are consistent with observations from SHIP-1-deficient mice, which express more myeloid suppressor cells than their WT counterparts and accept allogeneic bone marrow grafts with a reduced incidence of graft-versus-host disease (Ghansah et al., 2004; Kerr, 2008). In addition SHIP-1-null mice are better able to accept bone marrow transplants compared with control mice (Wang et al., 2002), and SHIP-1 deficient mice have shown reduced cardiac graft rejection compared with control mice (Paraiso et al., 2007). The inhibition of SHIP-1 using pharmacological compounds may therefore offer the potential to aid transplant acceptance in patients undergoing transplant surgery. SHIP-1 inhibitors increased levels of granulocytes, red blood cells, neutrophils, and platelets in mice and could therefore have potential to improve blood cell number in patients with myelodysplastic syndrome or myelosuppressive infection. 3AC also triggered the apoptosis of human acute myeloid leukemia cell lines, consistent with the antiapoptotic nature of SHIP-1 under some circumstances.

These SHIP-1-targeting small molecules offer interesting therapeutic opportunities. However, SHIP-1 can have either inhibitory or activating roles in cell signaling that are determined by whether signaling pathways distal to PI3K are promoted by the SHIP-1 substrate PI(3,4,5)P₃ or product PI(3,4)P₂ (Harris et al., 2008; Kerr, 2011). Moreover, SHIP-1 product and substrate can both influence Akt activation and cell survival. This may explain in part why both activators and inhibitors of SHIP-1 have shown efficacy against leukemic cells (Kerr, 2011). As with PI3K, the targeting of SHIP-1 with activators or inhibitors is not without its risks. SHIP-1 deficiency leads to Crohn's disease-like ileitis in mice, most likely as a result of defects in mucosal T-cell immunity (Kerr et al., 2011). Moreover, in addition to a core catalytic domain responsible for the hydrolysis of the 5′-phosphate on PI(3,4,5)P₃, SHIP-1 encodes multiple structural domains (SH2 and proline-rich regions as well as NPXY motifs that become tyrosine-phosphorylated and bind the phosphotyrosine-binding domain motif) that facilitate interaction with partner proteins (Harris et al., 2008). Targeting the catalytic activity of SHIP-1 may be beneficial because it will avoid any scaffolding roles of SHIP-1. Conversely, if the scaffolding role of SHIP-1 contributes to the pathological outcomes, such small molecules would be predicted to be ineffective.

Inhibition of SHIP-2, which shares protein domains and 35% homology with SHIP-1 (Fig. 1) has also been explored, because this lipid phosphatase has been implicated in diabetes and obesity (Ooms et al., 2009). Owing to its ubiquitous expression, SHIP-2 is expressed along with SHIP-1 in leukocytes and in diseases in which these two isoforms perform nearly or completely redundant functions, so pharmacological targeting of both isoforms may be preferential. Indeed, targeting of multiple myeloma cells by compounds that target both SHIP-1 and -2 can cause cell death (Fuhler et al., 2012). Compounds that selectively target SHIP-2 have also been reported, including inhibitors of SHIP-2 catalytic activity (Suwa et al., 2009) and a novel cell-impermeant biphenyl 2,3′4,5′,6-pentakisphosphate (Vandeput et al., 2007). Despite the lack of cell penetration by this latter compound, it has provided an invaluable tool for the study of SHIP-2. Indeed, the solving of the crystal structure of biphenyl 2,3′4,5′,6-pentakisphosphate revealed that upon binding of this compound to the phosphatase domain of SHIP-2, a flexible loop folds over and encloses the ligand (Vandeput et al., 2007; Mills et al., 2012). Targeting of this conformational change in the structure of SHIP-2 could present a novel feature for selectively targeting this lipid phosphatase. To fully exploit the potential of SHIP-1 and SHIP-2 inhibition for the treatment of inflammatory, autoimmune disorders and malignancies, much more needs to be learned about the role of SHIP-2 in immune cell function.