Selective Phosphatidylinositol 3-kinase Inhibitor Research Articles

Leucine‐rich glioma inactivated 3 promotes HaCaT keratinocyte migration

Our finding that human skin expresses leucine-rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3β (GSK3β) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3-kinase inhibitor, and LiCl, a selective GSK3β inhibitor, abolished LGI3-induced cell migration. We tested β-catenin levels after LGI3 treatment because the Akt-GSK3β pathway regulates β-catenin accumulation, and β-catenin promotes cell migration. LGI3 treatment increased β-catenin protein and nuclear localization, whereas LY294002 prevented LGI3-induced focal adhesion kinase and Akt activation as well as β-catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following β-catenin accumulation through the Akt pathway.

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.MethodsWe conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.ResultsThe phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.ConclusionsWe developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Ceramide 1-phosphate stimulates glucose uptake in macrophages

It is well established that ceramide 1-phosphate (C1P) is mitogenic and antiapoptotic, and that it is implicated in the regulation of macrophage migration. These activities require high energy levels to be available in cells. Macrophages obtain most of their energy from glucose. In this work, we demonstrate that C1P enhances glucose uptake in RAW264.7 macrophages. The major glucose transporter involved in this action was found to be GLUT 3, as determined by measuring its translocation from the cytosol to the plasma membrane. C1P-stimulated glucose uptake was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3K) or Akt, also known as protein kinase B (PKB), and by specific siRNAs to silence the genes encoding for these kinases. C1P-stimulated glucose uptake was also inhibited by pertussis toxin (PTX) and by the siRNA that inhibited GLUT 3 expression. C1P increased the affinity of the glucose transporter for its substrate, and enhanced glucose metabolism to produce ATP. The latter action was also inhibited by PI3K- and Akt-selective inhibitors, PTX, or by specific siRNAs to inhibit GLUT 3 expression.

Abstract P4-07-01: The Class I Selective PI3K Inhibitor GDC-0941 Enhances the Efficacy of Docetaxel in Human Breast Cancer Models by Increasing the Rate of Apoptosis

Abstract Docetaxel (DTX) is commonly used as a front-line treatment option for breast cancer but many patients ultimately relapse and succumb to disease progression. Phosphatidylinositol 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration and survival. Given that phosphatidylinositol 3-kinase (PI3K) is frequently activated in breast cancer and induces chemo-resistance, it is an attractive target for combination therapy with standard of care drugs such as DTX. The current study was intended to determine if GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the anti-tumor activity of DTX in human breast cancer models in vitro and in vivo. A panel of 25 breast tumor cell lines representing luminal, HER2+ and basal sub-types were treated with GDC-0941, DTX or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers and apoptosis induction. The combination of GDC-0941 and DTX decreased the cellular viability of breast tumor cell lines in vitro but with variable drug synergy. The addition of both drugs resulted in stronger synergistic effects in a sub-set of tumor cell lines that was not predicted by breast cancer sub-type or mutations within the PI3K pathway. Human xenografts of breast cancer cell lines and patient-derived tumors were utilized to assess efficacy of GDC-0941 and DTX in vivo. In these models, the best combination efficacy was detected when the two drugs were dosed within 1 hour of each other. We also observed that GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon co-treatment with DTX. Our data provides a preclinical rationale for evaluating GDC-0941 in combination with DTX for breast cancer treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-01.

PI3Kγ activation is required for LPS-induced reactive oxygen species generation in respiratory epithelial cells

In this study, we investigated the molecular basis of reactive oxygen species (ROS) generation induced by lipopolysaccharide (LPS) in A549 cells--an alveolar epithelial cell line. A549 cells or normal human bronchial epithelial (NHBE) cells were stimulated with LPS. ROS generation was measured in A549 cells or NHBE cells pre-treated with a selective inhibitor of phosphatidylinositol 3-kinase γ (PI3Kγ), AS 605240, PI3Kγ siRNA, or a ROS scavenger, pyridoxamine (PM). Treatment of A549 cells or NHBE cells with LPS caused a significant increase in intracellular ROS generation. Pretreatment with the PI3Kγ inhibitor, AS 605240 decreased the LPS-induced increase of ROS generation, phosphorylation of Akt, and production of phosphatidyl 3,4,5-trisphosphate in A549 cells. In addition, interference with siRNA for PI3Kγ significantly reduced LPS-induced ROS generation in A549 cells. Treatment of A549 cells with LPS or hydrogen peroxide increased the nuclear factor-κB (NF-κB) in the nucleus, accompanying an increase in phosphorylation of inhibitory κB-α, degradation of the protein, and reduction of cytosolic NF-κB. Pretreatment with AS 605240 reduced these LPS-induced changes. In addition, pretreatment with PM or N-acetyl cysteine resulted in inhibition of nuclear NF-κB activation. These results suggest that PI3Kγ plays a key role in LPS-induced ROS generation in alveolar epithelial cells, thereby activating NF-κB.

Electroacupuncture confers beneficial effects through ionotropic glutamate receptors involving phosphatidylinositol-3 kinase/Akt signaling pathway in focal cerebral ischemia in rats

Aim of the studyWe investigated the molecular mechanisms underlying the beneficial effects of electroacupuncture (EA) to cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model. Materials and methodsEA stimulation (2Hz, 1mA) was needle-delivered for 30min at acupoints corresponding to Baihui (GV20) and Qihai (CV6) in men in MCAO rats and was analyzed by an infarct volume, Western blot and immunohistochemical assay. ResultsEA stimulations strongly reduced infarct volume and improved neurological outcome after stroke. When we focused on the glutamate-evoked excitotoxic injury with coupled pathways, N-methyl-d-aspartate receptor (NMDAR) NR2A and NR2B and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR2 subunit phosphorylation were significantly arrested by EA treatment in the parietal cortex of the brain. On the contrary, phosphatidylinositol-3 kinase (PI3K) and Akt phosphorylation markedly increased. Treatment of MCAO rats with the selective PI3K inhibitor LY-294002 to confirm the signaling pathway involved in EA neuroprotection revealed abrogated neuroprotective effects induced by EA. ConclusionThese results suggest that the beneficial effects of EA were associated with reduction of NMDAR and AMPAR phosphorylation and with activation of PI3K/Akt pathway. The PI3K/Akt pathway may mainly contribute to EA-induced neuroprotection after stroke.

Preclinical Assessment of the Absorption and Disposition of the Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor GDC-0980 and Prediction of Its Pharmacokinetics and Efficacy in Human

(S)-1-{4-[2-(2-Amino-pyrimidin-5-yl)-7-methyl-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl]-piperazin-1-yl}-2-hydroxy-propan-1-one (GDC-0980) is a potent and selective inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin, two key components of the PI3K pathway, the deregulation of which is associated with the development of many cancers. The objectives of these studies were to characterize the absorption and disposition of GDC-0980 and assess its efficacy in an MCF7-neo/HER2 human breast cancer xenograft model in immunocompromised mice. Studies in parental Madin-Darby canine kidney cells indicated that GDC-0980 had high permeability (P(app) = 18 × 10⁻⁶ cm/s), suggesting good absorption potential. However, it was found to be a P-glycoprotein and breast cancer resistance protein substrate in transfected cells and in knockout mice studies. Plasma protein binding was low, with the fraction unbound ranging from 29 to 52% across species. GDC-0980 hepatic clearance (CL) was predicted to be low in all of the species tested from hepatocyte incubations. The plasma CL of GDC-0980 was low in mouse (6.30 ml · min⁻¹ · kg⁻¹), rat (15.4 ml · min⁻¹ · kg⁻¹), and dog (6.37 ml · min⁻¹ · kg⁻¹) and moderate in cynomolgus monkey (18.9 ml · min⁻¹ · kg⁻¹). Oral bioavailability ranged from 14.4% in monkey to 125% in dog. Predicted human plasma CL and volume of distribution using allometry were 5.1 ml · min⁻¹ · kg⁻¹ and 1.8 l/kg, respectively. Parameters estimated from the pharmacokinetic/pharmacodynamic modeling of the MCF7-neo/HER2 xenograft data indicated that the GDC-0980 plasma concentration required for tumor stasis was approximately 0.5 μM. These parameters, combined with the predicted human pharmacokinetic profile, suggested that 55 mg once daily may be a clinically efficacious dose. GDC-0980 preclinical characterization and the predictions of its human properties supported its clinical development; it is currently in Phase II clinical trials.

Abstract A43: The Pi3K inhibitor GSK2126458 is highly toxic in breast ductal carcinoma

Abstract Introduction: Breast carcinoma has been shown to harbor mutations that up-regulate phosphatidylinositol 3 kinase (PI3K) signaling pathway. Thus, targeting this pathway is expected to impair tumor survival. The aim of this work was to investigate the cytotoxicity of GSK2126458 (a highly potent and selective inhibitor of PI3K) in breast ductal carcinoma from a patient. Methods: A breast ductal carcinoma was collected immediately following resection. Tumor sections (0.5×0.5×0.1 cm) were immersed in ice-cold RPMI medium gassed with 95% O2:5% CO2. The samples were incubated at 37oC in RPMI (continuously gassed as above) with and without 50 nM GSK2126458 (Selleck Chemicals, cat. #S2658) for 90 min (final volume = 2.0 mL). At the end of the incubation period, 37 μM Ac-DEVD-AMC (a caspase-3 substrate) was added with and without the pan-caspase inhibitor zVAD-fmk (32.1 μM, added 10 min before the substrate). The reactions were allowed to continue at 37oC for 20 min before tissue processing for histology, immunohistochemistry for cleaved caspase -3 (Cell Signaling Technology, kit #8120), cellular respiration and caspase activation. Mitochondrial O2 consumption was determined as function of time from the phosphorescence decay rate (1/T) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. For caspase activity, the Ac-DEVD-AMC cleavage reaction was quenched with tissue disruption and passages through a 27-G needle. The supernatants were collected by centrifugation (∼16,300g for 90 min) through Microcentrifuge Filter (nominal molecular weight limit = 10,000 Dalton, Sigma©), separated on HPLC, and analyzed for the free fluorogenic AMC moiety. Results: This 73-year-old female had invasive ductal carcinoma (grade 3), measuring 4.5 cm. The tumor expressed estrogen and Her2-neu receptors. Ki-67 score was 70%. Compared to untreated tumor, the treated tumor revealed cellular disintegration with loss of cohesion, cytoplasmic vacuolization and numerous apoptotic bodies. Positive cleaved caspase-3 staining was noted in 10% of untreated tumor and 20% in treated tumor. The initial rate of mitochondrial O2 consumption (kc, in μM O2 min−1 mg−1) was 0.17. At 2 h, the value of kc for untreated tumor was 0.019 and for treated tumor 0.033. The AMC peak area (arbitrary units, reflecting intracellular caspase activation) in untreated tumor was 460,886, in tumor treated with GSK2126458 was 7,234,911 (15.7 fold increment), and in tumor treated with GSK2126458 plus zVAD-fmk was 1,523,682 (79% inhibition), confirming the cleavage of Ac-DEVD-AMC was mainly mediated by caspases. Conclusions: GSK2126458 induced remarkable necrosis in invasive ductal carcinoma. This preliminarily observation supports the role of PI3K activation in breast carcinogenesis and possible therapeutic utility of PI3K inhibitors in the treatment of invasive ductal carcinoma. The described in vitro methodology provides a rapid assessment of the cytotoxicity of PI3K/PTEN/Akt/mTOR inhibitors.

PI3K/Akt signaling pathway-induced heme oxygenase-1 upregulation mediates the adaptive cytoprotection of hydrogen peroxide preconditioning against oxidative injury in PC12 cells

Both the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and heme oxygenase-1 (HO-1) create a survival signal against oxidative stress-induced injuries. Although we have demonstrated that hydrogen peroxide (H2O2) preconditioning confers adaptive cytoprotection against oxidative stress-induced injury in PC12 cells, it remains unknown whether these defense systems are involved in the protective effect of H2O2 preconditioning. In the current study, PC12 cells were preconditioned with 100 µM H2O2 for 90 min, followed by 24 h recovery and subsequent exposure to 300 µM H2O2 for further 12 h. The findings showed that preconditioning with 100 µM H2O2 upregulated HO-1 expression. Zinc protoporphyrin IX (ZnPP), a selective inhibitor of HO-1, at a concentration of 15 µM, significantly attenuated H2O2 preconditioning-elicited cytotoxicity, apoptosis, oxidative stress and mitochondrial membrane potential (ΔΨm) loss in PC12 cells. In addition, H2O2 preconditioning enhanced phosphorylation of Akt. Treatment with 25 µM LY294002, a selective inhibitor of PI3K, for 20 min before H2O2 preconditioning blocked not only H2O2 preconditioning-induced HO-1 induction, but also the protective effect of H2O2 preconditioning against cytotoxicity. The present study provides novel evidence for the effect of preconditioning with H2O2 on the induction of HO-1, which contributes to the adaptive cytoprotection of H2O2 preconditioning against oxidative stress-induced cellular injury via a PI3K/Akt-dependent mechanism in PC12 cells.

Sevoflurane postconditioning involves an up-regulation of HIF-1α and HO-1 expression via PI3K/Akt pathway in a rat model of focal cerebral ischemia

Administration of sevoflurane at the onset of reperfusion has been confirmed to provide a cerebral protection. However, little is known about the mechanism. In this study, we tested the hypothesis that sevoflurane postconditioning induces neuroprotection through the up-regulation hypoxia inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1) involving phosphatidylinositol-3-kinase (PI3K)/Akt pathway. In the first experiment, male Sprague–Dawley rats were subjected to focal cerebral ischemia. Postconditioning was performed by exposure to 2.5% sevoflurane immediately at the onset of reperfusion. The mRNA and protein expression of HIF-1α and its target gene, HO-1, intact neurons and the activity of caspase-3 was evaluated at 6, 24 and 72h after reperfusion. In the second experiment, we investigated the relationship between PI3K/Akt pathway and the expression of HIF-1α and HO-1 in the neuroprotection induced by sevoflurane. Cerebral infarct volume, apoptotic neuron and the expression of HIF-1α, HO-1 and p-Akt were evaluated at 24h after reperfusion. Compared with the control group, sevoflurane postconditiong significantly ameliorated neuronal injury, up-regulated mRNA and protein levels of HIF-1α and HO-1, inhibited the activity of caspase-3, and decreased the number of TUNEL-positive cells and infarct sizes. However, the selective PI3K inhibitor, wortmannin not only partly eliminated the neuroprotection of sevoflurane as shown by reducing infarct size and apoptotic neuronal cells, but also reversed the elevation of HIF-1α, HO-1 and p-Akt expression in the ischemic penumbra induced by sevoflurane. Therefore, our data demonstrate that the cerebral protection from sevoflurane postconditioning is partly mediated by PI3K/Akt pathway via the up-regulation of HIF-1α and HO-1.

Synthesis and structure–activity relationships of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones as novel series of potent β isoform selective phosphatidylinositol 3-kinase inhibitors

A series of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved isoform selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Several compounds in the series demonstrated potent (∼ 0.100μM IC50) growth inhibition in a PTEN deficient cancer cell line.

Arg972 insulin receptor substrate-1 enhances tumor necrosis factor-α-induced apoptosis in osteoblasts.

The presence of Arg972 insulin receptor substrate-1 (IRS-1) is associated with impaired insulin/IRS-1 signaling to activate phosphatidylinositol-3 kinase (PI3K). Tumor necrosis factor-α (TNF-α), an inflammatory cytokine with a central role in the pathogenesis of rheumatoid arthritis (RA), induces apoptosis in osteoblasts, which are the principal cell type responsible for bone loss in RA. In our previous study, an association between Arg972 IRS-1 and a high risk and severity of RA was identified. In the present study, the effects of Arg972 IRS-1 and IRS-1 on TNF-α-induced apoptosis in human osteoblasts were examined. Normal and RA osteoblasts were stably transfected with Arg972 IRS-1 and IRS-1. In addition, cells were stably transduced with IRS-1-shRNA to knock down IRS1. Following stimulation with 10 nM insulin for 30 min, the stable overexpression of Arg972 IRS-1 and knock down of IRS-1 significantly decreased IRS-1-associated PI3K activity and Akt activation/phosphorylation at serine 473 (ser473) and enhanced TNF-α-induced apoptosis in normal and in RA osteoblasts. By contrast, the stable overexpression of IRS-1 significantly increased the levels of IRS-1-associated PI3K activity and Akt phosphorylation (ser473) and inhibited TNF-α-induced apoptosis, which was eliminated by pretreatment with 50 µn BJM120, a selective PI3K inhibitor, for 30 min. In conclusion, the present study provided the first evidence, to the best of our knowledge, that insulin stimulation of Arg972 IRS-1 and IRS-1 enhanced and inhibited TNF-α-induced apoptosis, respectively in normal and RA osteoblasts by a PI3K‑dependent mechanism. These findings suggest that insulin/IRS-1 signaling is important in the pathogenesis of RA.

Synthesis and structure–activity relationships of imidazo[1,2-a]pyrimidin-5(1H)-ones as a novel series of beta isoform selective phosphatidylinositol 3-kinase inhibitors

A series of PI3K-beta selective inhibitors, imidazo[1,2-a]-pyrimidin-5(1H)-ones, has been rationally designed based on the docking model of the more potent R enantiomer of TGX-221, identified by a chiral separation, in a PI3K-beta homology model. Synthesis and SAR of this novel chemotype are described. Several compounds in the series demonstrated potent growth inhibition in a PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage independent conditions.

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sjögren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sjögren's syndrome.

Antitumor Activity of NVP-BKM120—A Selective Pan Class I PI3 Kinase Inhibitor Showed Differential Forms of Cell Death Based on p53 Status of Glioma Cells

The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo. The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model. We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts). Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment.

Preclinical pharmacokinetics of the novel PI3K inhibitor GDC-0941 and prediction of its pharmacokinetics and efficacy in human

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregulation is associated with the development of many cancers.GDC-0941, a potent and selective inhibitor of PI3K, was characterised preclinically in in vitro and in vivo studies.Plasma protein binding was extensive, with free fraction less than 7%, and blood-to-plasma ratio ranged from 0.6 to 1.2 among the species tested. GDC-0941 human hepatic clearance was predicted to be moderate by liver microsomal incubations. GDC-0941 had high permeability in Madin-Darby canine kidney cells.The clearance of GDC-0941 was high in mouse (63.7 mL/min/kg), rat (49.3 mL/min/kg) and cynomolgus monkey (58.6 mL/min/kg), and moderate in dog (11.9 mL/min/kg). The volume of distribution ranged from 2.52 L/kg in rat to 2.94 L/kg in monkey. Oral bioavailability ranged from 18.6% in monkey to 77.9% in mouse.Predicted human clearance and volume of distribution using allometry were 6 mL/min/kg and 2.9 L/kg, respectively. The human efficacious doses were predicted based on results from preclinical pharmacokinetic studies and xenograft models.GDC-0941 preclinical characterisation and predictions of its properties in human supported its progression towards clinical development. GDC-0941 is currently in phase II clinical trials.

Abstract 4479: Targeting non-small cell lung cancer cells harboring a PIK3CA mutation by a novel and oral PI3K selective inhibitor, PF-4989216

Abstract Aberrant phosphatidylinositol 3-kinase (PI3K)/AKT signaling occurs commonly in cancer. Gene mutation, amplification, and copy number gains of the PI3K catalytic subunit p110α have been shown in a variety of human cancers. The tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) dephosphorylates the 3-phosphoinositides and is frequently mutated, deleted, or down-regulated in many cancers to constitutively activate the PI3K pathway. In response to activation, PI3Ks phosphorylate membrane phosphatidylinositides to generate phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 serves as an secondary messenger activating AKT and 3’-phosphoinositide-dependent kinase-1 (PDK1). Phosphorylation of AKT at threonine-308 (T308) by PDK1 and serine-473 (S473) by mTORC2 fully activates the AKT pathway to regulate various cellular processes including cell growth, proliferation, survival, and metabolism. Several inhibitors targeting the PI3K pathway have been developed in preclinical discovery or clinical trials; however, a small molecule inhibitor has not yet been approved for cancer treatment. Most inhibitors have inhibitory activity against both PI3Ks and mTOR kinases. The mTORC1 and mTORC2 complexes control critical pathways regulating cell proliferation, apoptosis, angiogenesis, and metabolism through AKT-dependent and AKT-independent mechanisms. Therefore, agents targeting PI3K with additional mTOR kinase inhibitory activities may carry extra toxicity through the disruption of mTOR function in normal cells. To develop a drug selectively targeting PI3K, we have identified PF-4989216 as a novel potent and selective PI3K inhibitor. In this study, we describe the in vitro and in vivo anti-tumor activity of PF-4989216 in human non-small-cell lung cancer cells (NSCLCs). PF-4989216 inhibits the phosphorylation of PI3K downstream molecules and subsequently leads to inhibition in cell proliferation and xenograft tumor growth in NSCLCs harboring a PIK3CA mutation, including an erlotinib resistant line, NCI-H1975. Although PF-4989216 inhibits the phosphorylation of PI3K downstream molecules in the NSCLCs with PTEN loss (NCI-H1650), PF-4989216 does not sufficiently induce anti-tumor efficacy in NCI-H1650. Our results suggest that there may be a different tumorigenesis mechanism between PIK3CA mutation and PTEN loss in NSCLCs, and indicate that PF-4989216 is a potential cancer drug candidate for erlotinib resistant non-small-cell lung cancer patients with PIK3CA mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4479. doi:10.1158/1538-7445.AM2011-4479

Abstract 3140: Analysis of candidate phosphatidylinositol 3’ kinase inhibitor biomarkers in matched synchronous primary and metastatic breast cancer tumors

Abstract The class I phosphatidylinositol 3’kinase (PI3K) is activated in a wide variety of human malignancies and inhibitors targeting the PI3K pathway hold great promise in the treatment and management of cancer. Successful development of such inhibitors will be enhanced by identification of responsive patients through the use of predictive biomarkers. Using cell lines and xenograft tumor models to evaluate a collection of putative biomarkers predictive of response to the selective inhibitor GDC-0941 in breast cancer, we have found excellent activity of the PI3K inhibitor GDC-0941 in luminal and HER2 amplified models, and that PIK3CA mutations and HER2 amplification are highly specific biomarkers of response to this agent. PTEN was predictive in some but not all cases, suggesting a need for further biomarkers of response in PTEN-driven disease. Here we report expanded analysis of human breast tumor samples, including matched primary tumor and lymph node metastases from 59 patients, suggesting primary and metastatic sites were generally concordant in terms of status of key biomarkers such as PTEN and PIK3CA, though several notable exceptions were discovered. We also compared PTEN status determined by immunohistochemistry (IHC) with a chromosomal fluorescence in situ hybridization (FISH) assay in a subset of tumors and found evidence for chromosomal deletion of the PTEN locus in several ER+ breast cancers, suggesting that some but not all of the PTEN loss observed in breast occurs at the chromosomal level and that both FISH and IHC may have utility as diagnostic assays. While we report that PIK3CA mutations and PTEN loss have reasonable prevalence to be considered as biomarkers for GDC-0941 in ER+ breast cancer, we have found a number of breast cancer models with no apparent pathway alterations but sensitivity to GDC-0941, indicating the need for additional predictive biomarkers. We identified a collection of genes for which expression levels indicate PI3K pathway activation and correlate with in vitro sensitivity to GDC-0941 as well as apparent prognostic significance in retrospective analysis of breast tumors. We report here expression analysis of these genes across matched primary breast tumors and lymph node metastases. In summary, our data provide strong rationale for developing a selective PI3K inhibitor in the ER+ breast cancer setting, and a framework for clinical implementation of predictive biomarker assays with potential utility in patient selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2011-3140

Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin‐dependent control of epithelial Na+ absorption

Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention. We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins. Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases. While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1.

LY294002 may overcome 5-FU resistance via down-regulation of activated p-AKT in Epstein-Barr virus-positive gastric cancer cells

BackgroundAs EBV-associated gastric cancer has unique features that are different from EBV (-) gastric cancer, EBV is considered to have a key role in gastric carcinogenesis. It has been reported that viral latent membrane protein 2A (LMP2A) in EBV-transformed tumor cells activates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which provides a survival signal and chemo-resistance to cytotoxic anti-cancer drugs. This study was to evaluate anti-proliferative effect and cell cycle change when 5-FU and LY294002 (LY), a selective inhibitor of PI3K, were treated separately or combined with different schedules in EBV positive gastric cancer cell line, SNU-719.MethodsAfter single treatment and sequential combination of 5-FU and LY, cytotoxic activity was measured by MTS assay. When 5-FU and LY were treated in single and sequential combinations, the expression of p-AKT, p-NFkB, p-p53 and bcl-2 was observed on different concentrations by Western blot analysis. We also investigated the effect on apoptosis and cell cycle distribution using flow cytometry. The LMP2A siRNA inhibition was done to confirm the reversal of decreased 5-FU activity and p-AKT.ResultsWhen 5-FU was sequentially combined with LY, the combination index (CI) value indicated synergistic anti-proliferative effect. The expression of p-AKT and p-NFκB was upregulated by 5-FU alone but sequential treatment of 5-FU and LY decreased the expression of both p-AKT and p-NFκB. When 5-FU was combined with LY, G0/G1 and sub G1 cell population (%) increased. When 5-FU was added to the cells transfected with LMP2A siRNA, its anti-proliferative effect increased and the expression of p-AKT decreased. In sequential combination of 5-FU and LY, the expression of p-p53 was increased and bcl-2 expression was diminished compared to 5-FU alone.ConclusionThese data suggest that sequential combination of 5-FU and LY induce synergistic cytotoxicity and overcome intrinsic and acquired resistance of 5-FU via downregulation of activated p-AKT and mitochondria-dependent apoptosis in EBV gastric cancer cell line, SNU-719.