Constitutive Activation Of Phosphatidylinositol 3-kinase Research Articles

Inhibition of PI3K promotes anti-tumor immune responses to Mantle Cell Lymphoma

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma that is characterized by a chromosomal translocation resulting in overexpression of Cyclin D1, dysregulation of the cell cycle, and constitutive activation of phosphatidylinositol 3 kinase (PI3K). MCL patients also have decreased natural killer T (NKT) cells – an innate-like lymphocyte population involved in initiating the anti-tumor immune response. We hypothesize that constitutive activation of PI3K in MCL alters CD1d-mediated antigen processing and presentation, ultimately resulting in decreased anti-tumor NKT cell responses. To investigate the role of PI3K on MCL survival and immunogenicity in vitro and in vivo, we utilized a panel of PI3K inhibitors (PI3Ki). Pretreatment of human MCL cell lines with micromolar concentrations of PI3Ki resulted in increased apoptosis and enhanced immunogenicity. While dual PI3K/mTOR inhibitor Dactolisib (BEZ-235), completely abrogates NKT/T cell responses of in vitro, stimulated peripheral blood mononuclear cells, PI3K-specific inhibition allows immune cells to function. In a murine model of MCL, PI3Ki-treated animals had decreased tumor burden and increased NKT/T cell responses to ex vivo stimulation with the most significant effect seen in the Buparlisib (BKM-120)-treated group. To elucidate the mechanism, MCL cells were treated in vitro with BKM-120 and flow cytometric analysis indicated increased CD1d expression, but decreased MHC class I expression. These results suggest that selectively targeting PI3K can reduce toxicity, as well as have a significant impact on tumor burden and immune function.

Abstract 159: Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530

Abstract Introduction: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a fundamental role in many cellular processes like growth, survival, proliferation, differentiation and motility. In cancers several mutations have been identified that lead to constitutive activation of PI3K. PQR530 is a novel, ATP site directed inhibitor of all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 that is currently in pre-clinical development. PQR530 potently binds to its targets, inhibits cell proliferation and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR530 inhibits PI3K signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been found for PQR530 during GLP toxicological testing in rats and dogs. Increase in insulin and blood glucose, a treatable class effect of PI3K inhibitors, has been observed after PQR530 administration to mice. Investigation of mutagenicity and hERG binding resulted in a clean profile. PQR530 exhibited dose-proportional pharmacokinetics (PK) in male C57BL/6J mice. A maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (7.8 μg/ml and 112.6 μg/ml, respectively) indicating that efficacious concentrations were reached in both tissues. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. PQR530 potently inhibited PI3K signaling in vivo for several hours after administration of a single oral dose of 50 mg/kg. Tumor growth was significantly decreased in SUDHL-6 lymphoma, RIVA lymphoma and OVCAR-3 ovarian cancer mouse xenografts using daily, oral administration. Conclusion: PQR530 is a potent, ATP competitive pan-PI3K and mTORC1/2 inhibitor. The physico-chemical properties of PQR530 result in good oral bioavailability and excellent brain penetration. PQR530 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Rageot D, et al., Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor, abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Petra Hillmann, Denise Rageot, Florent Beaufils, Anna Melone, Alexander Sele, Robert A. Ettlin, Jürgen Mestan, Vladimir Cmiljanovic, Marc Lang, Elisabeth Singer, Carolin Walter, Hoa HP Nguyen, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2017-159

The role of PI3K in modulating NKT cell responses to Mantle Cell Lymphoma

Abstract Natural Killer T (NKT) cells are important in the initiation and regulation of immune responses to cancers. The observation that mantle cell lymphoma (MCL) patients have a significant reduction in both NKT cell number and function suggests that the antitumor effects mediated by these cells may be compromised. MCL is characterized by the chromosomal translocation and nuclear overexpression of Cyclin D1. Secondary chromosomal abnormalities and dysregulation of cell cycle machinery also contribute to MCL pathogenesis, such as aberrant or constitutive activation of phosphatidylinositol 3 kinase (PI3K). We hypothesize that constitutive activation of PI3K in MCL alters the repertoire of lipids in these cells, ultimately resulting in decreased CD1d-mediated NKT responses to MCL. In this study, we used small molecule pharmacologic inhibitors of PI3K (PI3Ki) to investigate the role of PI3K signaling on MCL survival and immunogenicity both in vitro and in vivo. Treatment with PI3Ki induced apoptosis in human MCL cell lines and enhanced their immunogenicity. In addition, we have found that treatment of peripheral blood mononuclear cells with isoform-specific PI3Ki does not suppress T cell activation; whereas, treatment with a pan PI3Ki, BEZ-235, completely abrogates NKT/T cell responses. These studies suggest inhibitors targeting individual isoforms may have higher therapeutic efficacy. Collectively, this work will advance our understanding of differences in the modes of PI3K signaling in MCL survival and in antigen processing and presentation.

An Open-Label, Multicenter, 2-Arm, Dose-Finding, Phase 1b Study of the Combination of Ruxolitinib and Buparlisib (BKM120) in Patients with Myelofibrosis: Results from HARMONY Study

An Open-Label, Multicenter, 2-Arm, Dose-Finding, Phase 1b Study of the Combination of Ruxolitinib and Buparlisib (BKM120) in Patients with Myelofibrosis: Results from HARMONY Study

A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes.

IntroductionThe activation of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is one the most frequent genetic events in breast cancer, consequently the development of PI3K inhibitors has attracted much attention. Here we evaluate the effect of PI3K inhibition on global gene expression in breast cancer cells.MethodsWe used a range of methodologies that include in silico compound analysis, in vitro kinase assays, cell invasion assays, proliferation assays, genome-wide transcription studies (Agilent Technologies full genome arrays), gene set enrichment analysis, quantitative real-time PCR, immunoblotting in addition to chromatin immunoprecipitation.ResultsWe defined the physico-chemical and the biological properties of ETP-45658, a novel potent PI3K inhibitor. We demonstrated that ETP-45658 potently inhibited cell proliferation within a broad range of human cancer cells, most potently suppressing the growth of breast cancer cells via inhibiting cell cycle. We show that this response is Forkhead box O (FOXO) protein dependent and p53 independent. Our genome-wide microarray analysis revealed that the cell cycle was the most affected biological process after exposure to ETP-45658 (or our control PI3K inhibitor PI-103), that despite the multiple transcription factors that are regulated by the PI3K/AKT signalling cascade, only the binding sites for FOXO transcription factors were significantly enriched and only a subset of all FOXO-dependent genes were induced. This disparity in gene transcription was not due to differential FOXO promoter recruitment.ConclusionsThe constitutive activation of PI3Ks and thus the exclusion of FOXO transcription factors from the nucleus is a key feature of breast cancer. Our results presented here highlight that PI3K inhibition activates specific FOXO-dependent genes that mediate cell cycle arrest in breast cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0482-y) contains supplementary material, which is available to authorized users.

Hijacking Dlg1 for oncogenic phosphatidylinositol 3-kinase activation in human epithelial cells is a conserved mechanism of human adenovirus E4-ORF1 proteins.

The E4-ORF1 gene of human adenoviruses encodes a 14-kDa protein that promotes viral replication as well as cellular metabolic reprogramming, survival, and transformation by constitutively activating cellular phosphatidylinositol 3-kinase (PI3K). We recently reported that the E4-ORF1 protein from subgroup D human adenovirus type 9 upregulates and oncogenically activates PI3K by a novel mechanism involving separate interactions of E4-ORF1 with cellular discs large 1 (Dlg1) and PI3K to form a ternary complex that translocates to the plasma membrane (K. Kong, M. Kumar, M. Taruishi, and R. T. Javier, PLoS Pathog. 10:e1004102, 2014, doi:10.1371/journal.ppat.1004102). The current study was carried out to investigate whether other human adenovirus E4-ORF1 proteins share this mechanism of action. The results showed that in human MCF10A epithelial cells, stable expression of E4-ORF1 proteins encoded by representative human adenovirus serotypes from subgroups A to D induce ternary complex formation, Dlg1-dependent PI3K activation, PI3K protein elevation, Dlg1 and PI3K membrane recruitment, and PI3K-dependent cellular transformation. The first three of these E4-ORF1 activities were also observed in MCF10A cells infected with each wild-type human adenovirus from subgroups A to D. Our findings indicate that most, if not all, human adenovirus E4-ORF1 proteins share a conserved molecular mechanism of PI3K activation, which confers a common capacity to promote oncogenic transformation in human epithelial cells. PI3K activation by the adenovirus E4-ORF1 protein mediates oncogenic cellular transformation by human adenovirus type 9, augments viral protein expression and replication by human adenovirus type 5, and dysregulates cellular glucose and lipid metabolism by human adenovirus type 36. For the first time, we report that E4-ORF1 proteins from human adenoviruses in subgroups A to D evolved a conserved molecular mechanism to mediate constitutive PI3K activation that can provoke oncogenic transformation in human epithelial cells. The results raise potential safety concerns about the use of vectors encoding the E4-ORF1 gene in human gene therapy and vaccination. Our findings further suggest that the conserved mechanism revealed here may be targeted for development of therapeutic drugs to treat and prevent adenovirus-associated human diseases.

Sirt2 Deacetylase Is a Novel AKT Binding Partner Critical for AKT Activation by Insulin

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr(308) and Ser(473), mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis. Here, we identify the cytoplasmic NAD(+)-dependent deacetylase, Sirt2, as a novel AKT interactor, required for optimal AKT activation. Pharmacological inhibition or genetic down-regulation of Sirt2 diminished AKT activation in insulin and growth factor-responsive cells, whereas Sirt2 overexpression enhanced the activation of AKT and its downstream targets. AKT was prebound with Sirt2 in serum or glucose-deprived cells, and the complex dissociated following insulin treatment. The binding was mediated by the pleckstrin homology and the kinase domains of AKT and was dependent on AMP-activated kinase. This regulation involved a novel AMP-activated kinase-dependent Sirt2 phosphorylation at Thr(101). In cells with constitutive PI3K activation, we found that AKT also associated with a nuclear sirtuin, Sirt1; however, inhibition of PI3K resulted in dissociation from Sirt1 and increased association with Sirt2. Sirt1 and Sirt2 inhibitors additively inhibited the constitutive AKT activity in these cells. Our results suggest potential usefulness of Sirt1 and Sirt2 inhibitors in the treatment of cancer cells with up-regulated PI3K activity and of Sirt2 activators in the treatment of insulin-resistant metabolic disorders.

GW24-e1260 Overexpressing cellular repressor of E1A-stimulated genes promote hypoxia-induced VEGF paracrine via HIF-1 in mesenchymal stem cells

ObjectivesHypoxia inducible factor-1α (HIF-1α) is the key transcription regulator for multiple angiogenic factors, including vascular endothelial growth factor (VEGF). Cellular repressor of E1A-stimulated genes (CREG) has also been identified a...

Obesity and Thyroid Cancer: Is Leptin the (Only) Link?

Obesity and Thyroid Cancer: Is Leptin the (Only) Link?

ASSA13-10-9 Overexpressing Cellular Repressor of E1A-Stimulated Genes Promote Hypoxia-Induced VEGF Paracrine Via HIF-1α in Mesenchymal Stem Cells

BackgroundHypoxia inducible factor-1α (HIF-1α) is the key transcription regulator for multiple angiogenic factors, including vascular endothelial growth factor (VEGF). Cellular repressor of E1A-stimulated genes (CREG) has also been identified a...

Plausible role of naringenin against cerebrally implanted C6 glioma cells in rats

Gliomas encompass a significant percentage of intrinsic neoplasms of the central nervous system in both adults and children. The constitutive activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B is the hallmark of glioma. The up-regulated protein kinase B could influence the expression of cyclooxygenase-2, an indicator of aggressive glioma. The present study was embarked to demonstrate the effect of naringenin (50 mg/kg bw for 30 days administrated orally) on PI3K, protein kinase B, and cyclooxygenase-2 in cerebrally implanted rat C6 glioma model. After the experimental period of 30 days, the animals were sacrificed and excised brain tissues were subjected to study the expressions of PI3K, protein kinase B, and cyclooxygenase-2 by reverse transcriptase polymerase chain reaction followed Western blot analysis. The activity of COX-2 (production of prostaglandin-E(2)) was also determined by high pressure liquid chromatography. The results showed that the naringenin could down-regulate the expressions of PI3K and protein kinase B along with activity and expression of cyclooxygenase-2 in C6 glioma cells implanted rat brain. In conclusion, it can be argued that the reduced expressions of phosphatidylinositol 3-kinase and protein kinase B in naringenin-treated glioma-induced rat brain might be involved in the down-regulation of cyclooxygenase-2 expression and activity. Thus, fine-tuned investigation of which will be helpful for targeted drug discovery against glioma.

Cardiac PI3K-Akt Impairs Insulin-Stimulated Glucose Uptake Independent of mTORC1 and GLUT4 Translocation

Impaired insulin-mediated glucose uptake characterizes cardiac muscle in humans and animals with insulin resistance and diabetes, despite preserved or enhanced phosphatidylinositol 3-kinase (PI3K) and the serine-threonine kinase, Akt-signaling, via mechanisms that are incompletely understood. One potential mechanism is PI3K- and Akt-mediated activation of mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K), which may impair insulin-mediated activation of insulin receptor substrate (IRS)1/2 via inhibitory serine phosphorylation or proteasomal degradation. To gain mechanistic insights by which constitutive activation of PI3K or Akt may desensitize insulin-mediated glucose uptake in cardiomyocytes, we examined mice with cardiomyocyte-restricted, constitutive or inducible overexpression of a constitutively activated PI3K or a myristoylated Akt1 (myrAkt1) transgene that also expressed a myc-epitope-tagged glucose transporter type 4 protein (GLUT4). Although short-term activation of PI3K and myrAkt1 increased mTOR and S6 signaling, there was no impairment in insulin-mediated activation of IRS1/2. However, insulin-mediated glucose uptake was reduced by 50-80%. Although longer-term activation of Akt reduced IRS2 protein content via an mTORC1-mediated mechanism, treatment of transgenic mice with rapamycin failed to restore insulin-mediated glucose uptake, despite restoring IRS2. Transgenic activation of Akt and insulin-stimulation of myrAkt1 transgenic cardiomyocytes increased sarcolemmal insertion of myc-GLUT4 to levels equivalent to that observed in insulin-stimulated wild-type controls. Despite preserved GLUT4 translocation, glucose uptake was not elevated by the presence of constitutive activation of PI3K and Akt. Hexokinase II activity was preserved in myrAkt1 hearts. Thus, constitutive activation of PI3K and Akt in cardiomyocytes impairs GLUT4-mediated glucose uptake via mechanisms that impair the function of GLUT4 after its plasma-membrane insertion.

Autophagy restricts proliferation driven by oncogenic phosphatidylinositol 3-kinase in three-dimensional culture

Autophagy is a tightly regulated lysosomal self-digestion process that can both promote and impede tumorigenesis. Here, we utilize a three-dimensional (3D) culture model to address how interactions between autophagy and the PI3K/Akt/mTOR pathway impact the malignant behavior of cells carrying a tumor-derived, activating mutation in PI3K (PI3K-H1047R). In this model, autophagy simultaneously mediates tumor suppressive and promoting functions within individual glandular structures. In 3D culture, constitutive PI3K activation overcomes proliferation arrest and promotes resistance to anoikis in the luminal space, resulting in aberrant structures with filled lumen. Inhibiting autophagy in PI3K-H1047R structures triggers luminal cell apoptosis, resulting in lumen clearance. At the same time, ATG depletion strongly enhances PI3K-H1047R cell proliferation during 3D morphogenesis, revealing an unexpected role for autophagy in restricting proliferation driven by PI3K activation. Intriguingly, over-expression of the autophagy cargo receptor p62/SQSTM1 in PI3K-H1047R cells is sufficient to enhance cell proliferation, activate the ERK/MAPK pathway, and to promote EGF-independent proliferation in 3D culture. Overall, these results indicate that autophagy antagonizes specific aspects of oncogenic PI3K transformation, with the loss of autophagy promoting proliferation.

Ginsenoside-Rg1 mediates a hypoxia-independent upregulation of hypoxia-inducible factor-1α to promote angiogenesis

Hypoxia-inducible factor (HIF-1) is the key transcription regulator for multiple angiogenic factors and is an appealing target. Ginsenoside-Rg1, a nontoxic saponin isolated from the rhizome of Panax ginseng, exhibits potent proangiogenic activity and has the potential to be developed as a new angiotherapeutic agent. However, the mechanisms by which Rg1 promotes angiogenesis are not fully understood. Here, we show that Rg1 is an effective stimulator of HIF-1α under normal cellular oxygen conditions in human umbilical vein endothelial cells. HIF-1α steady-state mRNA was not affected by Rg1. Rather, HIF-1α protein synthesis was stimulated by Rg1. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector p70 S6 kinase (p70S6K), but not extracellular-signal regulated kinase 1/2. We further revealed that HIF-1α induction triggered the expression of target genes, including vascular endothelial growth factor (VEGF). The use of small molecule inhibitors LY294002 or rapamycin to inhibit PI3K/Akt and p70S6K activities, respectively, resulted in diminished HIF-1α activation and subsequent VEGF expression. RNA interference-mediated knockdown of HIF-1α suppressed Rg1-induced VEGF synthesis and angiogenic tube formation, confirming that the effect was HIF-1α specific. Similarly, the angiogenic phenotype could be reversed by inhibition of PI3K/Akt and p70S6K. These results define a hypoxia-independent activation of HIF-1α, uncovering a novel mechanism for Rg1 that could play a major role in angiogenesis and vascular remodeling.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-011-9235-z) contains supplementary material, which is available to authorized users.

Posttranscriptional regulation of the p85α.alpha; adapter subunit of phosphatidylinositol 3-kinase in human leukemia cells

Constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling has been observed in up to 70% of acute myeloid leukemia. Class IA PI3K consists of a catalytic subunit (p110α, p110β, p110δ) and an adapter subunit (p85α, p55α, p50α, p85β, p55γ). The p85α adapter subunit stabilizes the catalytic p110 subunit and recruits p110 to the plasma membrane. In addition, p85α inhibits the basal activity of p110α and can negatively regulate signal transduction, as shown for insulin and GM-CSF receptor signaling. Here, we describe that the expression of p85α is posttranscriptionally regulated in several human and murine leukemia cell lines and in a Hodgkin lymphoma cell line (CO) by translational repression. A detailed analysis of CO cells revealed that both wild type and a mutated p85α mRNA are detectable at similar ratios in the nucleus and polysomes. However, while the mutated p85α protein is expressed in CO cells, translation of the wild type p85α mRNA is completely inhibited. Ectopic expression of wild type p85α from a retroviral vector is suppressed in CO cells and in five out of six leukemia cell lines. Our data indicate that leukemia cells can regulate the expression of p85α by posttranscriptional regulation.

Approaches and limitations of phosphatidylinositol-3-kinase pathway activation status as a predictive biomarker in the clinical development of targeted therapy

The central role played by the class I(A) phosphatidylinositol-3-kinase (PI3K) signaling node in human cancer is highlighted in the multiple mechanisms by which these signals become dysregulated. Many studies suggest that constitutive PI3K activation in human cancer contributes to drug resistance, including targeted agents and standard cytotoxic therapy. The combination of activation mechanisms and the multiple downstream cascades that emanate from the PI3K node contributes to the difficulty in measuring PI3K activation as a biomarker. Although many agents suppress the pathway in models, the challenge remains to translate this biology into a patient selection strategy (i.e., identify patients with "PI3K activated" tumors) and subsequently link this biomarker definition to drug responses in patients. The various genetic and epigenetic lesions resulting in pathway activation necessitate combined approaches using genetic, genomic, and protein biomarkers to accurately characterize "PI3K activated" tumors. Such a combined approach to pathway status can be assessed using a statistical stratification of patients in a randomized trial into "pathway on" and "pathway off" subsets to compare the treatment effect in each arm. Instead of considering individual biomarkers for their predictive ability, this strategy proposes the use of a collection of biomarkers to identify a specific "pathway on" patient population predicted to have clinical benefit from a pathway inhibitor. Here, we review the current understanding of the mechanisms of PI3K activation in breast cancer and discuss a pathway-based approach using PI3K as a predictive biomarker in clinical development, which is currently in use in a global phase 3 setting.

DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis

In metastatic prostate cancer (PCa) cells, imbalance between cell survival and death signals such as constitutive activation of phosphatidylinositol 3-kinase (PI3K)-Akt and inactivation of apoptosis-stimulated kinase (ASK1)-JNK pathways is often detected. Here, we show that DAB2IP protein, often down-regulated in PCa, is a potent growth inhibitor by inducing G(0)/G(1) cell cycle arrest and is proapoptotic in response to stress. Gain of function study showed that DAB2IP can suppress the PI3K-Akt pathway and enhance ASK1 activation leading to cell apoptosis, whereas loss of DAB2IP expression resulted in PI3K-Akt activation and ASK1-JNK inactivation leading to accelerated PCa growth in vivo. Moreover, glandular epithelia from DAB2IP(-/-) animal exhibited hyperplasia and apoptotic defect. Structural functional analyses of DAB2IP protein indicate that both proline-rich (PR) and PERIOD-like (PER) domains, in addition to the critical role of C2 domain in ASK1 activity, are important for modulating PI3K-Akt activity. Thus, DAB2IP is a scaffold protein capable of bridging both survival and death signal molecules, which implies its role in maintaining cell homeostasis.

PI3 Kinase Inhibition Induces Apoptotic Cell Death in Acute Lymphoblastic Leukemia Via the Mitochondrial Pathway.

The phosphatidylinositol 3-kinase (PI3K) pathway regulates many cellular processes that are involved in tumor progression. Aberrant activation of the PI3K pathway due to an alteration of its elements like PTEN or Akt occurs quite frequently in malignant cells. Thus, inhibition of this pathway represents a promising option for the treatment of cancer patients. The best characterized PI3K inhibitors are LY294002 and wortmannin that were shown to disrupt downstream signaling and induce apoptotic cell death in tumor cells. Acute lymphoblastic leukemia (ALL) is a malignancy mainly found in young children and elderly with constitutive activation of PI3K pathway.In our study we analyzed the effect of PI3K inhibition in cell lines deduced from ALL (Jurkat, BV173, SD1, T-2) and primary leukemic cells by incubating them with increasing concentrations of inhibitors of the PI3K signaling. We found that treatment of ALL cells with LY294002, the mTOR inhibitor rapamycin or Akt inhibitor SH5 induced apoptotic cell death that was accompanied by caspase-3 activation and PARP-cleavage and interfered with intracellular PI3K/Akt signaling as analyzed by phosphorylation and expression of mTOR or P70S6K. In line with these results apoptotic cell death could be inhibited by the pan-caspase inhibitor zVAD. In order to determine the pathway of apoptosis induction we took advantage of Jurkat cells (T-ALL) overexpressing or lacking molecules involved in apoptotic pathways such as FADD, an adaptor molecule recruited to the death receptor upon ligand binding, Caspase-8, Caspase-9 or Bcl-2, an anti-apoptotic protein that prevents the release of cytochrom c from mitochondria. PI3K inhibition by LY294002 induced apoptotic cell death in cells deficient of FADD or caspase-8 with no difference to wild type cells. In contrast, cells overexpressing Bcl2 or lacking caspase-9 were resistant to apoptotic death indicating that PI3-kinase inhibition is independent of the external death receptor signaling and is mediated via the mitochondrial pathway. These results were confirmed by analyzing PARP cleavage and caspase-3 activation in utilized leukemic cell lines. Furthermore, we found that the PI3K inhibitor LY294002 induced apoptosis in ALL cells that could be increased by the etoposide, a topoisomerase inhibitor, or TRAIL. In addition, in contrast to etoposide, treatment of ALL cells with TRAIL could overcome the resistance of ALL cells to PI3K inhibition even in caspase-9 deficient Jurkat cells. Our results provide an interesting approach in designing novel therapeutic strategies to target the PI3K pathway in ALL to overcome the resistance to cytotoxic agents.

Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin‐induced cell death

The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is well known for the regulation of cell survival, proliferation, and some metabolic routes. In this study, we document a novel role for the PI3-K/Akt pathway during cell death induced by apoptin, a tumour-selective inducer of apoptosis. We show for the first time that apoptin interacts with the p85 regulatory subunit, leading to constitutive activation of PI3-K. The inhibition of PI3-K activation either by chemical inhibitors or by genetic approaches severely impairs cell death induced by apoptin. Downstream of PI3-K, Akt is activated and translocated to the nucleus together with apoptin. Direct interaction between apoptin and Akt is documented. Co-expression of nuclear Akt significantly potentiates cell death induced by apoptin. Thus, apoptin-facilitated nuclear Akt, in contrast to when in its cytoplasmic pool, appears to be a positive regulator, rather than repressor of apoptosis. Our observations indicate that PI3-K/Akt pathways have a dual role in both survival and cell death processes depending on the stimulus. Nuclear Akt acts as apoptosis stimulator rather than as a repressor, as it likely gains access to a new set of substrates in the nucleus. The implicated link between survival and cell death pathways during apoptosis opens new pharmacological opportunities to modulate apoptosis in cancer, for example through the manipulation of Akt's cellular localization.

A Conserved Role for Phosphatidylinositol 3-Kinase but Not Akt Signaling in Mitochondrial Adaptations that Accompany Physiological Cardiac Hypertrophy

Physiological cardiac hypertrophy is associated with mitochondrial adaptations that are characterized by activation of PGC-1alpha and increased fatty acid oxidative (FAO) capacity. It is widely accepted that phosphatidylinositol 3-kinase (PI3K) signaling to Akt1 is required for physiological cardiac growth. However, the signaling pathways that coordinate physiological hypertrophy and metabolic remodeling are incompletely understood. We show here that activation of PI3K is sufficient to increase myocardial FAO capacity and that inhibition of PI3K signaling prevents mitochondrial adaptations in response to physiological hypertrophic stimuli despite increased expression of PGC-1alpha. We also show that activation of the downstream kinase Akt is not required for the mitochondrial adaptations that are secondary to PI3K activation. Thus, in physiological cardiac growth, PI3K is an integrator of cellular growth and metabolic remodeling. Although PI3K signaling to Akt1 is required for cellular growth, Akt-independent pathways mediate the accompanying mitochondrial adaptations.