P110β Isoform Research Articles

Rules of engagement: distinct functions for the four class I PI3K catalytic isoforms in immunity

Mammalian cells can express up to four different class I phosphatidylinositide 3-kinase (PI3K) isoforms, each of which is engaged by tyrosine kinases or G protein-coupled receptors (GPCRs) to generate the second messenger signaling molecule PtdIns(3,4,5)P₃ (PIP₃). The p110α and p110β isoforms are relatively widely expressed, whereas p110γ and p110δ are more highly expressed in cells of the immune system than in other cell types. Nevertheless, each of the four class I PI3Ks have been shown to participate in the orchestration of the signaling events that lead to immune cell development and control of gene expression, skewing toward individual cell lineage subsets and proliferation.

Allosteric activation of PI3Kα by oncogenic mutations

The gene encoding the p110α catalytic subunit of class IA phosphoinositide 3-kinase (PIK3CA) is one of the most frequently mutated genes in human cancer [1]. Somatic mutations are widely dispersed throughout every domain of the catalytic subunit (Fig ​(Fig1A).1A). Two “hot spot” mutants have been shown biochemically and structurally to activate lipid kinase activity by distinct mechanisms [2]. Rare mutations, which have been shown to play a role in endometrial cancers [3], also increase lipid kinase activity. The mechanism of activation of rare mutations that are distant from both the active site and membrane interface have remained ambiguous. We have recently determined a set of allosteric conformational events that occur upon activation of wild type PI3Kα using hydrogen/deuterium exchange mass spectrometry (HDX-MS), and we find that all oncogenic mutants studied activated lipid kinase activity by mimicking or enhancing these specific events [4]. Figure 1 Oncogenic mutants in PI3K The lipid kinase activity of the class IA PI3Ks is tightly regulated by the p85 regulatory subunit which stabilizes, inhibits, and allows activation downstream of receptor tyrosine kinases (RTKs) (Fig. 1A, 1B). The interaction between the catalytic and regulatory subunit is mediated by the interaction of the adaptor-binding domain (ABD) of p110 and the coiled coil iSH2 domain of p85 [5]. The enzyme is inhibited by contacts between the N-terminal SH2 domain (nSH2) of p85 and the C2, helical and lipid kinase domains of the p110 subunit [5, 6]. The binding of phosphorylated tyrosine residues in RTKs and their associated proteins by the nSH2 disrupts this inhibitory interface and activates the enzyme. The enzyme is also inhibited by contacts between the C2 of p110 and the iSH2 of p85 [7]. The C-terminal SH2 (cSH2) domain of p85 which inhibits the p110δ and p110β isoforms of PI3K has no inhibitory influence on the p110α isoform [8]. To determine the mechanism of activation by oncogenic mutants we first investigated conformational changes that occur in the wild type upon binding to tyrosine-phosphorylated peptides derived from RTKs (pY), and upon binding to phospholipid membranes [4]. We identified four distinct conformational events that occur in the wild type PI3Kα upon activation (Fig. ​(Fig.1C):1C): (i) Disruption of the nSH2/helical interface, (ii) Disruption of the C2/iSH2 interface, (iii) Movement of the ABD relative to the kinase domain, and (iv) Interaction of the lipid kinase domain with membranes. We propose that these events are involved in the transition of a cytosolic, inhibited form of p110α to an open, activated form on the membrane surface. Oncogenic mutants in the ABD (G106V), ABD-RBD (Ras-binding domain) linker (G118D), C2 (N345K, C420R), and the hot spot mutants in the helical (E545K) and kinase domains (H1047R) upregulated lipid kinase activity by mimicking or enhancing one or more of the conformational events identified in the wild type. Mutations in the ABD and ABD-RBD linker caused both enhanced movement of the ABD domain relative to the kinase domain, and disruption of the C2/iSH2 interface compared to the WT enzyme. This same relationship was seen in mutations in the C2 domain, which also caused disruption of the C2/iSH2 interface along with movement of the ABD relative to the kinase domain. This shows that these two regions are allosterically linked to each other. These mutations can be activated by RTK derived pY, and are hyperactivated compared to the WT enzyme. The presence of the helical domain hotspot mutation located at the nSH2/helical interface fully disrupted this contact and made the mutant insensitive to pY activation, as predicted from previous biochemical and structural data [5, 6]. The kinase domain hotspot mutation caused conformational changes in the C-lobe of the kinase domain located at the membrane interface. Interestingly, the regions of the kinase domain that were exposed by this mutation are the exact same regions that were protected by the cSH2 domain in the p110β and p110δ isoforms [8]. This raises an interesting possibility that p110α has acquired inhibitory mutations in the kinase domain that enable it to bypass cSH2 mediated inhibition that occurs in the p110β and p110δ isoforms. The important role of oncogenic mutants of PI3Kα in the development of a variety of human cancers makes the development of novel inhibitory strategies an important goal. The discovery of these unexpected allosteric conformational changes that are enhanced in oncogenic mutants opens up a novel strategy towards designing small molecule inhibitors that function outside of the conserved ATP binding pocket.

Mechanisms of PI3Kβ-Selective Inhibition Revealed by Reciprocal Mutagenesis

The p110β isoform of PI3 kinase (PI3Kβ) has been implicated in pathological disorders such as thrombosis and cancer and a number of PI3Kβ-selective inhibitors have recently progressed into clinical studies. Although crystallography studies identify a binding site conformation favored by the inhibitors, no specific interaction explains the observed selectivity. Using site-directed mutagenesis we have identified a specific tyrosine residue of the binding site Y778 that dictates the ability of the PI3Kβ isoform to bind these inhibitors. When mutated to isoleucine, PI3Kβ has reduced ability to present a specific cryptic binding site into which a range of reported PI3Kβ inhibitors can bind, and conversely when tyrosine is introduced into the same position in PI3Kα, the same inhibitors gain potency. The results provide a cogent explanation for the selectivity profiles displayed by these PI3K inhibitors and maybe others as well.

Enhancement of Tumor Cell Susceptibility to NK Cell Activity Through Inhibition of the PI3K Signaling Pathway.

Abstract 2152NK cells are the primary effectors of the innate immune response against cells that have undergone malignant transformation. Unlike T cells, NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the NK response against individual cells. Tumor cells have developed ways to escape killing by NK cells through a variety of mechanisms that are poorly understood. Using shRNA libraries, we previously demonstrated that several common signaling pathways modulate susceptibility of various tumor cells to NK cell activity. In this study we focused on one of the PI3K sub-units (PI3KCB, p110β isoform) that was identified in this genetic screen. The PI3K pathway has been linked to diverse cellular functions including cell growth, proliferation, survival and intracellular trafficking but PI3K signaling has not previously been associated with susceptibility to NK cells.To examine the mechanisms of PI3KCB involvement in tumor susceptibility to NK cell lysis, we tested 4 tumor cell lines representing different hematologic malignancies: IM9-multiple myeloma, U937-acute myeloid leukemia, K562-chronic myeloid leukemia and Jurkat-acute T cell leukemia. Stable cell lines were established expressing 4 independent shRNAs (P1, P2, P3, P4-shRNA) and 2 irrelevant shRNAs as controls. Western blot and RT-PCR showed different silencing activity of individual PI3KCB-shRNAs with efficient silencing of PI3KCB by 3 of 4 shRNAs in IM9 (P1, P2, P4-shRNA), 2 of 4 in K562 and U937 (P1, P2-shRNA) and 2 of 4 in Jurkat (P2, P4-shRNA). No PI3KCB gene silencing or protein down regulation was observed with P3-shRNA that was used as an additional negative control. PI3KCB silencing did not affect the proliferative capacity of the 4 tumor cell lines tested over a 5 day period. When compared with irrelevant shRNAs or P3-shRNA, effective silencing of PI3KCB in IM-9, K562 and U937 induced increased IFNγ secretion when target cells were incubated with primary purified NK effector cells and 2 different human NK cell lines (NKL and NK-92). Silencing of PI3KCB in Jurkat target cells did not affect IFNγ secretion by NK cells. To determine whether increased IFNγ secretion by NK cells correlated with increased lytic activity, we incubated target cells with NK effectors overnight and assessed cytotoxicity using AnnexinV/7AAD. Lysis of IM9-PI3KCB-P1, P2 and P4-shRNA was increased by 10%-15% compared to irrelevant controls (P=0.04, P=0.04 and P=0.03 respectively); lysis of U937-PI3KCB-P1 and P2-shRNA was increased by 20% and 22% (P=0.02 and P=0.04 respectively); and lysis of K562-PI3KCB-P1 and P2-shRNA was increased by 10% and 20% (P=0.04 and P=0.04, respectively). Correlating with IFNγ assays, increased lysis was not detected for the Jurkat-PI3KCB-silenced cell lines.To define mechanisms responsible for increased susceptibility to NK cells, we examined the expression of several activating/inhibitory ligands on the membrane of PI3KCB-silenced tumor cells. Flow cytometry demonstrated increased expression of MICB (NKG2D ligand) and CD48 (2B4 ligand) in IM9-PI3KCB-KO lines; increased expression of MICA, MICB, CD48 and CD112/CD115 (DNAM-1 ligands) in K562-PI3KCB-KO lines; and increased expression of CD48, CD112 in U937-PI3KCB-KO lines. Reduced expression of MHC class I was also found in IM9 and U937 lines after PI3KCB silencing. Importantly, none of the activating/inhibitory ligands tested were modulated in Jurkat cells after PI3KCB silencing. Co-culture experiments with NK cells and blocking antibodies targeting NKG2D significantly reduced NK IFNγ secretion against IM9-PI3KCB-KO and K562-PI3KCB-KO targets when compared to controls. In contrast, DNAM-1 blocking in U937-PI3KCB-KO produced a uniform decrease of IFNγ secretion in U937-PI3KCB-KO and controls suggesting that in this target, modulation of other NK ligands could also be involved. These data demonstrate that the beta sub-unit of PI3K (PI3KCB) can modulate tumor susceptibility in different hematologic malignancies and that this effect is, at least in part, due to modulation of several activating/inhibitory ligands. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies. Disclosures:No relevant conflicts of interest to declare.

Discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-one-containing Phosphoinositide 3-kinase (PI3K) p110β isoform inhibitors through structure-based fragment optimisation

The discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-one-containing inhibitors of Phosphoinositide 3-kinases (PI3K) p110β isoform is reported. Structure-based optimisation of the original fragment hit resulted in lead compounds with improvements in ligand efficiency, lipophilicity efficiency, p110β potency and selectivity over p110α.

Discovery of phosphoinositide 3-kinases (PI3K) p110β isoform inhibitor 4-[2-hydroxyethyl(1-naphthylmethyl)amino]-6-[(2S)-2-methylmorpholin-4-yl]-1H-pyrimidin-2-one, an effective antithrombotic agent without associated bleeding and insulin resistance

Structure-based evolution of the original fragment leads resulted in the identification of 4-[2-hydroxyethyl(1-naphthylmethyl)amino]-6-[(2S)-2-methylmorpholin-4-yl]-1H-pyrimidin-2-one, (S)-21, a potent, selective phosphoinositide 3-kinases (PI3K) p110β isoform inhibitor with favourable in vivo antiplatelet effect. Despite its antiplatelet action, (S)-21 did not significantly increase bleeding time in dogs. Additionally, due to its enhanced selectivity over p110α, (S)-21 did not induce any insulin resistance in rats.

The p110α and p110β isoforms of PI3K play divergent roles in mammary gland development and tumorigenesis

Class Ia phosphatidylinositol 3 kinase (PI3K) is required for oncogenic receptor-mediated transformation; however, the individual roles of the two commonly expressed class Ia PI3K isoforms in oncogenic receptor signaling have not been elucidated in vivo. Here, we show that genetic ablation of p110α blocks tumor formation in both polyoma middle T antigen (MT) and HER2/Neu transgenic models of breast cancer. Surprisingly, p110β ablation results in both increased ductal branching and tumorigenesis. Biochemical analyses suggest a competition model in which the less active p110β competes with the more active p110α for receptor binding sites, thereby modulating the level of PI3K activity associated with activated receptors. Our findings demonstrate a novel p110β-based regulatory role in receptor-mediated PI3K activity and identify p110α as an important target for treatment of HER2-positive disease.

Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser(773), A-66S bound the region 1 non-conserved amino acid p110α Gln(859), and J-32 binding had an indirect interaction with Lys(776) and Ile(771). The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.

Functional Characterization of an Isoform-Selective Inhibitor of PI3K-p110β as a Potential Anticancer Agent

Genetic approaches have shown that the p110β isoform of class Ia phosphatidylinositol-3-kinase (PI3K) is essential for the growth of PTEN-null tumors. Thus, it is desirable to develop p110β-specific inhibitors for cancer therapy. Using a panel of PI3K isoform-specific cellular assays, we screened a collection of compounds possessing activities against kinases in the PI3K superfamily and identified a potent and selective p110β inhibitor: KIN-193. We show that KIN-193 is efficacious specifically in blocking AKT signaling and tumor growth that are dependent on p110β activation or PTEN loss. Broad profiling across a panel of 422 human tumor cell lines shows that the PTEN mutation status of cancer cells strongly correlates with their response to KIN-193. Together, our data provide the first pharmacologic evidence that PTEN-deficient tumors are dependent on p110β in animals and suggest that KIN-193 can be pursued as a drug to treat tumors that are dependent on p110β while sparing other PI3K isoforms. We report the first functional characterization of a p110β-selective inhibitor, KIN-193, that is efficacious as an antitumor agent in mice. We show that this class of inhibitor holds great promise as a pharmacologic agent that could be used to address the potential therapeutic benefit of treating p110β-dependent PTEN-deficient human tumors.

Beta-Testing of PI3-Kinase Inhibitors: Is Beta Better?

Although it has been known for some time that PTEN-null tumors require expression of the p110β isoform of phosphoinositide 3-kinase for growth, the corollary demonstration that small-molecule inhibitors of p110β are effective drugs for such tumors has not been shown. This has now been rectified by the demonstration that the TGX221 analogue KIN-193 is effective in mouse xenografts of HCC70 and PC3 human tumor cell lines.

Both p110α and p110β isoforms of PI3K can modulate the impact of loss-of-function of the PTEN tumour suppressor

The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN+/− mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P3, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN+/− mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P3 levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P3 levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.

Class-IA Phosphoinositide 3-Kinase p110β Triggers GPCR-Induced Superoxide Production in p110γ-Deficient Murine Neutrophils

Studies with knockout mice have indicated that the only isoform of phosphoinositide 3-kinase (PI3K) functioning in the oxidative burst of mouse neutrophils in response to heterotrimeric guanine nucleotide-binding protein–coupled receptor (GPCR) agonists is a class-IB PI3K, p110γ. In the present study, we observed that the cells from p110γ−/− mice gain a response to N-formyl-Met-Leu-Phe (fMLP) after priming with cytochalasin E. Even the unprimed cells, which show no response to fMLP, produce a significant amount of superoxide, when an effective agonist of the mouse-type fMLP receptors, Trp-Lys-Tyr-Met-Val-D-Met, is used to stimulate the cells. These results suggested that the class-IA isoforms (p110α, p110β, and p110δ) of PI3K are sufficient to trigger and maintain superoxide production. Examination of the effects of isoform-specific inhibitors suggested that the p110β isoform is the primary PI3K triggering the response to GPCR agonists when p110γ is absent.

PI3K Inhibition with GDC-0941 Has Greater Efficacy Compared to p110δ-Selective Inhibition with CAL-101 in Mantle Cell Lymphoma and May Be Particularly Advantageous in Multiply Relapsed Patients

Abstract 1654 Background:Mantle cell lymphoma (MCL) is an incurable, aggressive subtype of non-Hodgkin lymphoma in which there is a need for novel targeted therapies. Activation of the PI3K-Akt pathway and its role in the pathogenesis of MCL has been highlighted in a number of studies. Constitutive activation of the PI3K pathway inactivates GSK-3β, a downstream target of Akt, that can phosphorylate cyclin D1 resulting in its nuclear export. There is also evidence that cyclin D1 mRNA stability and translation is enhanced by this pathway. The class Ia PI3K p110 catalytic subunit isoforms α, β and δ are primarily implicated in oncogenesis. While the PI3K p110δ isoform is known to be enriched in lymphocytes, a gain of PIK3CA (the gene encoding PI3K p110α) copy number has been shown to be a frequent alteration in MCL. The expression and relative importance of the individual Class Ia PI3K isoforms has not been documented in this disease. With the development of isoform selective inhibitors, this is an important issue that needs to be addressed. Aims:We studied the expression of class Ia PI3K isoforms in primary MCL with relation to morphological variants and disease status. We also compared the efficacy of PI3K inhibition in MCL cell lines and primary samples using two novel inhibitors, GDC-0941(predominantly p110α/δ-selective) and CAL-101 (δ-selective), both of which are in early phase clinical trials. Methods:Tissue microarrays were constructed from triplicate 1mm cores from 144 MCL biopsies and 16 tonsil controls. The levels of p110α, p110β and p110δ isoforms were then determined by immunohistochemistry using isoform-specific antibodies. The in vitro effect of PI3K inhibitors on cell viability and apoptosis was studied in 4 MCL cell lines, (Jeko-1, Granta519, REC-1 and JVM-2), and 15 primary MCL samples. Expression of the class Ia PI3K isoforms and changes in downstream targets of PI3K were determined by western blotting. Results:P110δ was expressed at a consistently higher level in MCL samples and normal tonsil controls compared to the α and β isoforms, while p110β expression was weak and significantly lower than p110α expression. On comparing expression of isoforms at diagnosis and relapse, p110α expression was significantly increased beyond 1st relapse compared to diagnostic biopsies (p=0.04) and tonsil controls (p=0.02), an observation that was even more apparent in 6 paired samples [p=0.008, median IHC score 19.6 (5.0−53.2) at diagnosis vs. 91.5 (38.6 − 129) beyond 1st relapse]. No significant change was found in the expression of p110β or p110δ between diagnostic and relapse samples. There was no significant difference in expression levels of the 3 isoforms between blastoid and non-blastoid morphological variants. Expression of both the p110α and δ isoforms was detected by western blotting in 4 MCL cell lines, but only Jeko-1 cells were sensitive to inhibition with GDC-0941. CAL-101 produced little or no apoptosis in all 4 cell lines. In primary MCL samples, GDC-0941 was consistently more potent than CAL-101, with decrease in cell viability of 32 vs. 20% at 1μM (p=0.15), 51 vs. 25% at 5μM (p=0.02) and 67 vs. 35% at 10μM (p<0.0001) GDC-0941 and CAL-101 respectively. GDC-0941 was also able to partially overcome the stimulatory effect of sCD40L and IL4 on primary MCL samples. Western blotting showed a consistent reduction in the phosphorylation of Akt and GSK-3β in sensitive MCL cells. Conclusion:Our studies demonstrate that although p110δ is the most consistently expressed isoform, the expression of the p110α subunit increases significantly in multiply relapsed MCL. This observation, in combination with significantly greater in vitro sensitivity of MCL primary samples to GDC-0941, compared to the p110δ-selective inhibitor CAL-101, provides strong evidence for further evaluation of GDC-0941 in this disease. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria. Joel:Astra Zeneca: Research Funding; Intellikine: Research Funding.

Discovery of novel class 1 phosphatidylinositide 3-kinases (PI3K) fragment inhibitors through structure-based virtual screening

The discovery of ligand efficient and lipophilicity efficient fragment inhibitors of class 1 phosphatidylinositide 3-kinases (PI3K) is reported. A fragment version of the AstraZeneca compound bank was docked to a homology model of the PI3K p110β isoform. Interaction-based scoring of the predicted binding poses served to further prioritise the virtual fragment hits. Experimental screening confirmed potency for a total of 18 fragment inhibitors, belonging to five different structural classes.

The catalytic class IA PI3K isoforms play divergent roles in breast cancer cell migration

Transforming growth factor-β (TGFβ) plays an important role in breast cancer metastasis. Here phosphoinositide 3-kinase (PI3K) signalling was found to play an essential role in the enhanced migration capability of fibroblastoid cells (FibRas) derived from normal mammary epithelial cells (EpH4) by transduction of oncogenic Ras (EpRas) and TGFβ1. While expression of the PI3K isoform p110δ was down-regulated in FibRas cells, there was an increase in the expression of p110α and p110β in the fibroblastoid cells. The PI3K isoform p110β was found to specifically contribute to cell migration in FibRas cells, while p110α contributed to the response in EpH4, EpRas and FibRas cells. Akt, a downstream targets of PI3K signalling, had an inhibitory role in the migration of transformed breast cancer cells, while Rac, Cdc42 and the ribosomal protein S6 kinase (S6K) were necessary for the response. Together our data reveal a novel specific function of the PI3K isoform p110β in the migration of cells transformed by oncogenic H-Ras and TGF-β1.

A constitutively activated form of the p110β isoform of PI3-kinase induces prostatic intraepithelial neoplasia in mice

Recent work has shown that ablation of p110beta, but not p110alpha, markedly impairs tumorigenesis driven by loss of phosphatase and tensin homolog (PTEN) in the mouse prostate. Other laboratories have reported complementary data in human prostate tumor lines, suggesting that p110beta activation is necessary for tumorigenesis driven by PTEN loss. Given the multiple functions of PTEN, we wondered if p110beta activation also is sufficient for tumorigenesis. Here, we report that transgenic expression of a constitutively activated p110beta allele in the prostate drives prostate intraepithelial neoplasia formation. The resulting lesions are similar to, but are clearly distinct from, the ones arising from PTEN loss or Akt activation. Array analyses of transcription in multiple murine prostate tumor models featuring PI3K/AKT pathway activation allowed construction of a pathway signature that may be useful in predicting the prognosis of human prostate tumors.

Both p110α and p110β Isoforms of Phosphatidylinositol 3‐OH‐Kinase are Required for Insulin Signalling in the Hypothalamus

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3-kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross-talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform-selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague-Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110alpha and p110beta. Total hypothalamic PI3K activity was inhibited 65% by a p110alpha inhibitor, and 35% by a p110beta inhibitor, with a combination of inhibitors being equally effective as the broad-spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110alpha and p110beta inhibitors partly prevented insulin-induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre-treatment with a combination of p110alpha and p110beta inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre-treatment with the combination of p110alpha and p110beta inhibitors. The data suggest that p110alpha and p110beta isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin-dependent sensitisation of leptin action.

Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo

AbstractDuring platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110β null mouse model and demonstrated a critical role of PI3Kβ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glyco-protein VI-Fc receptor γ-chain complex, and its contribution in response to G-protein–coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110β null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein–coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3Kβ is also acting downstream of αIIbβ3. In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl3 injury of the carotid, suggesting that PI3Kβ is a potential target for antithrombotic drugs.

Isoform-Specific Phosphoinositide 3-Kinase Inhibitors Exert Distinct Effects in Solid Tumors

Therapeutic inhibitors are being developed against the phosphoinositide 3-kinase (PI3K) pathway, the deregulation of which drives tumor growth and survival in many cancers. There are eight PI3Ks in mammals divided into three classes. Class IA PI3Ks (p110alpha, p110beta, and p110delta) are critical for cell growth and survival, with the p110alpha isoform implicated as the most important in carcinomas. In this study, we examined the effects of small-molecule inhibitors of class IA PI3Ks to explore the contributions of different isoforms in cancer cells. Similar responses were seen in cancer cells with wild-type or activated mutant PI3K genes treated with p110alpha/delta or p110alpha/beta/delta inhibitors in cell viability assays. In contrast, PTEN-negative cell lines tended to be less responsive (4-fold overall) to an inhibitor of p110alpha/delta versus p110alpha/beta/delta. Combining a p110alpha/delta inhibitor with a p110beta inhibitor resulted in comparable potency to the p110alpha/beta/delta inhibitor. The disparity in efficacy was confirmed in vivo. Pharmacodynamic biomarker analysis revealed that an inhibitor with insufficient potency against the p110beta isoform was less effective at inhibiting the PI3K pathway in PTEN-negative tumor xenografts. Our results imply that patients with PTEN-negative tumors may preferentially benefit from treatment with a class I PI3K inhibitor that is capable of inhibiting the p110beta isoform.

Phosphoinositide 3-Kinase p110β Regulates Integrin αIIbβ3 Avidity and the Cellular Transmission of Contractile Forces

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.