Class IA Phosphoinositide 3-kinase Research Articles

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.

Class IA PI3K p110β Subunit Promotes Autophagy through Rab5 Small GTPase in Response to Growth Factor Limitation

Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its guanosine triphosphate (GTP)-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

Selective Inhibition of Phosphoinositide 3-Kinase p110α Preserves Lymphocyte Function*

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

PAQR3 Modulates Insulin Signaling by Shunting Phosphoinositide 3-Kinase p110α to the Golgi Apparatus

Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin signaling. We demonstrate here a new mode of spatial regulation for the p110α subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. PAQR3 interacts with p110α, and the intracellular targeting of p110α to the Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 overexpression. Insulin-stimulated PI3K activity and phosphoinositide (3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3β, but not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 interacts with the domain of p110α involved in its binding with p85, the regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin signaling by shunting cytosolic p110α to the Golgi apparatus while competing with p85 subunit in forming a PI3K complex with p110α.

Neuregulin1 Improved Cardiac Function in Doxorubicin-Treated Mice with Cardiomyocyte-Specific over expression of a Dominant-Negative PI3Kp110α

Neuregulin1s (NRG1s) are effective for protecting the heart from various stresses. Our previous studies show that heterozygous knockout of the NRG1 gene worsens while injections of a recombinant NRG1 improve cardiac function in Chemotherapy Drug Doxorubicin (DOX) - treated mice. In cultured cardiomyocytes, studies show that the Phosphoinositide 3-Kinase (PI3K) pathway is necessary for NRG1 to reduce DOX-induced apoptosis and loss of cardiac troponins. Here, we test whether PI3Kp110α, a Class IA PI3K isoform, is necessary for NRG1’s cardioprotective effects in DOX-treated mice. DOX was administered to mice with cardiomyocyte-specific overexpression of a constitutively active PI3Kp110α (CaPI3K), or a dominant negative PI3Kp110α (dnPI3K). Solvent or NRG1 was administered concurrently with DOX to dnPI3K mice. Our results showed that survival and cardiac function were improved in CaPI3K, but worsened in dnPI3K mice. Concurrent NRG1 injections significantly improved survival and cardiac function in dnPI3K mice, which were associated with increased activation of ERK1/2 and mTORC1 in the heart. These results have demonstrated that PI3Kp110α is important for protecting the heart from DOX. Further, the results suggest that PI3Kp110α is not necessary for NRG1 to protect the heart from DOX. NRG1 may protect the heart by activating alternative survival pathways, such as ERK1/2 and mTORC1, in DOX-treated dnPI3K mouse hearts.

Targeting the Phosphoinositide 3-Kinase p110-α Isoform Impairs Cell Proliferation, Survival, and Tumor Growth in Small Cell Lung Cancer

The phosphoinositide 3-kinase (PI3K) pathway is fundamental for cell proliferation and survival and is frequently altered and activated in neoplasia, including carcinomas of the lung. In this study, we investigated the potential of targeting the catalytic class I(A) PI3K isoforms in small cell lung cancer (SCLC), which is the most aggressive of all lung cancer types. The expression of PI3K isoforms in patient specimens was analyzed. The effects on SCLC cell survival and downstream signaling were determined following PI3K isoform inhibition by selective inhibitors or downregulation by siRNA. Overexpression of the PI3K isoforms p110-α and p110-β and the antiapoptotic protein Bcl-2 was shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K p110-α with RNA interference or selective pharmacologic inhibitors resulted in strongly affected cell proliferation of SCLC cells in vitro and in vivo, whereas targeting p110-β was less effective. Inhibition of p110-α also resulted in increased apoptosis and autophagy, which was accompanied by decreased phosphorylation of Akt and components of the mTOR pathway, such as the ribosomal S6 protein, and the eukaryotic translation initiation factor 4E-binding protein 1. A DNA microarray analysis revealed that p110-α inhibition profoundly affected the balance of pro- and antiapoptotic Bcl-2 family proteins. Finally, p110-α inhibition led to impaired SCLC tumor formation and vascularization in vivo. Together our data show the key involvement of the PI3K isoform p110-α in the regulation of multiple tumor-promoting processes in SCLC.

Increased Tonic PI3K Signaling Through p110α Can Limit the Efficacy of P110δ-Selective Inhibition in Mantle Cell Lymphoma, Particularly with Multiple Relapse

AbstractAbstract 1652 Background:Activation of the phosphoinositide-3 kinase (PI3K) pathway contributes to mantle cell lymphoma (MCL) pathogenesis, but early phase studies of the p110δ selective inhibitor GS-1101 demonstrate inferior responses in MCL compared to CLL and indolent NHL. The relative importance of the class Ia PI3K isoforms - p110α, p110β and p110δ in MCL is not clear. While p110δ is enriched in leucocytes, p110α gene amplification has been reported in 68% of MCL. Loss of PTEN expression occurs in approximately 15% of MCL and evidence in solid tumors suggests an essential role for p110β in PI3K signaling associated with PTEN loss. We previously reported significant up-regulation of p110α expression in relapsed disease as well as significantly greater in vitro cytotoxicity with GDC0941, a predominantly p110α/δ inhibitor, compared to the p110δ selective inhibitor GS-1101 in MCL. To investigate this further, we used isoform selective inhibitors to study the relative contribution of class Ia isoforms to PI3K signaling in the context of B-cell receptor (BCR) activation, p110α over-expression and PTEN deletion in this disease. We also screened tumor cells for activating mutations in PIK3CA and PIK3R1, the genes encoding p110α and p85α respectively. Methods:Peripheral blood mononuclear cells (PBMCs) and lymph nodes from MCL patients were used in addition to two MCL cell lines Jeko1 and Granta519. Goat anti-human IgM f(a’b) fragments were used for BCR stimulation. Immunohistochemistry (IHC) and western blotting were used to assess PTEN expression. In addition to GDC0941 and GS-1101, A66 was used for p110α-selective inhibition and TGX-221 for p110β inhibition. GS-1101 was purchased from Active Biochem and all other inhibitors from Selleck Chem. Changes in downstream targets of PI3K (p-Akt t308, p-Akt s473 and p-S6 s235/236) were evaluated using western blotting. DNA was extracted from 10 primary samples and 2 cell lines. Exons 9, 10, 11, 13, 15 and 16 of PIK3R1 and exons 9 and 20 of PIK3CA, were sequenced and screened for activating mutations. Results:p110δ is expressed at high levels in MCL and p110δ selective inhibition with GS-1101 was sufficient to block BCR-stimulated activation of the PI3K pathway in MCL cells. In order to investigate the relevance of increased p110α in relapsed MCL, that we previously reported, we treated the Granta519 MCL cell line, which exhibits p110α gene amplification and constitutive Akt phosphorylation, with GS-1101 and GDC0941. In GS-1101 treated cells, phosphorylation of Akt was still detectable at low levels after 2 hours of treatment while p-Akt was undetectable in cells treated with equimolar concentrations (1μM) of GDC0941. At 24 hours, GS-1101 treated cells showed an increase in Akt phosphorylation compared to 2 hour levels suggesting ongoing p110α mediated signaling despite p110δ inhibition, whereas p-Akt remained undetectable in GDC-0941 treated cells. These changes were reflected in downstream phosphorylation of ribosomal S6. The addition of the highly selective p110β inhibitor TGX221 to GS-1101 did not have any additional effect on p-Akt whereas adding a p110α selective inhibitor (A66) mimicked the activity of GDC0941, further supporting the role of p110α in p110δ-independent PI3K signaling. We found loss of PTEN expression in 17% (25/147) of MCL biopsies by IHC. There was no change in the pattern of class Ia isoform expression in tumors with loss of PTEN and adding a p110β inhibitor to GS-1101 did not increase cytotoxicity in MCL primaries that had loss of PTEN expression. No activating PIK3CA or PIK3R1 mutations were detected in 10 primary MCL samples and 2 cell lines. Conclusion:In healthy B-cells, p110δ is essential for BCR mediated PI3K signaling but both p110α and p110δ contribute to tonic PI3K signaling. Our studies confirm a similar essential role for p110δ in MCL cells both by its expression and its role in BCR signaling. However, over-expression of p110α in MCL, especially in relapsed disease, increases the contribution of this isoform to tonic PI3K signaling thereby limiting the efficacy of p110δ-selective inhibition. The increased expression of p110α with relapse may reflect reduced dependence of tumor cells on survival signals from their microenvironment. We conclude that whereas PI3K p110β does not appear to play a significant role in MCL, inhibition of both p110α and δ isoforms appears essential for effective PI3K inhibition in this disease. Disclosures:Auer:Pharmacyclics: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Class I PI3K-mediated Akt and ERK signals play a critical role in FcεRI-induced degranulation in mast cells

Class IA and IB phosphoinositide 3-kinases (PI3Ks) have been shown to regulate mast cell functions such as proliferation, development, survival and degranulation, but the functional redundancy between these two PI3K signaling pathways in mast cells remains unclear. Here, we have generated mice deficient in both class IA regulatory subunit p85α and class IB catalytic subunit p110γ, and show that p85α(-/-)p110γ(-/-) mice exhibit a more severe defect in mast cell development than single-knockout mice. In addition, the in vivo passive cutaneous anaphylaxis reaction of p85α(-/-)p110γ(-/-) mice was nearly completely abrogated, whereas single-knockout mice exhibit just marginal reduction. Pharmacological inactivation of Akt in wild-type bone marrow-derived mast cells (BMMCs) led to partial reduction of degranulation, while over-expression of a constitutively active Akt partially restored the impaired degranulation in p85α(-/-)p110γ(-/-) BMMCs. We also found that the extracellular signal-regulated kinase (ERK) signaling pathway was activated in a PI3K-dependent manner upon FcεRI stimulation and that simultaneous inhibition of Akt and ERK resulted in nearly complete blockade of FcεRI-induced degranulation. Our data provide evidence that Akt and ERK pathways play redundant roles in FcεRI-induced degranulation.

Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis

Class I(A) phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP(3)). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP(3) generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.

Dynamic steps in receptor tyrosine kinase mediated activation of class IA phosphoinositide 3-kinases (PI3K) captured by H/D exchange (HDX-MS)

The catalytic subunits of all class IA phosphoinositide 3-kinases (PI3Ks) associate with identical p85-related subunits and phosphorylate PIP2 yielding PIP3, but they can vary greatly in the signaling pathways in which they participate. The binding of the p85 subunit to the p110 catalytic subunits is constitutive, and this inhibits activity, but some of the inhibitory contacts are reversible and subject to regulation. Interaction with phosphotyrosine-containing peptides (RTK-pY) releases a subset of these inhibitory contacts. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) provides a map of the dynamic interactions unique to each of the isotypes. RTK-pY binding exposes the p110 helical domains for all class IA enzymes (due to release of the nSH2 contact) and exposes the C-lobe of the kinase domains of p110β and p110δ (resulting from release of the cSH2 contact). Consistent with this, our in vitro assays show that all class IA isoforms are inhibited by the nSH2, but only p110β and p110δ are inhibited by the cSH2. While a C2/iSH2 inhibitory contact exists in all isoforms, HDX indicates that p110β releases this contact most readily. The unique dynamic relationships of the different p110 isozymes to the p85 subunit may facilitate new strategies for specific inhibitors of the PI3Ks.

Isoform-selective induction of human p110δ PI3K expression by TNFα: identification of a new and inducible PIK3CD promoter

PI3Ks (phosphoinositide 3-kinases) are signalling molecules and drug targets with important biological functions, yet the regulation of PI3K gene expression is poorly understood. Key PI3Ks are the class IA PI3Ks that consist of a catalytic subunit (p110α, p110β and p110δ) in complex with a p85 regulatory subunit. Whereas p110α and p110β are ubiquitously expressed, high levels of p110δ are mainly found in white blood cells, with most non-leucocytes expressing low levels of p110δ. In the present paper we report that TNFα (tumour necrosis factor α) stimulation induces p110δ expression in human ECs (endothelial cells) and synovial fibroblasts, but not in leucocytes, through transcription start sites located in a novel promoter region in the p110δ gene (PIK3CD). This promoter is used in all cell types, including solid tumour cell lines that express p110δ, and is activated by TNFα in ECs and synovial fibroblasts. We further present a detailed biochemical and bioinformatic characterization of p110δ gene regulation, demonstrating that PIK3CD has distinct promoters, some of which can be dynamically activated by pro-inflammatory mediators. This is the first molecular identification of a PI3K promoter under the control of acute extracellular stimulation.

Down-regulation of p110β expression increases chemosensitivity of colon cancer cell lines to oxaliplatin

This study examined the synergetic effect of class IA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-V FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G(0)-G(1) phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wild-type group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.

PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

PI3K and mTOR Signaling Pathways in Cancer: New Data on Targeted Therapies

The mammalian target of rapamycin (mTOR) and the phosphoinositide 3-kinase (PI3K) signaling pathways are commonly deregulated in cancers and promote cellular growth, proliferation, and survival. mTOR is part of two complexes, mTORC1 and mTORC2, with different biochemical structures and substrates specificity. PI3K/AKT activation may result from genetic hits affecting different components of the pathway, whereas the mechanisms leading to constitutive mTORC1 activation remain globally unknown. The connections between the PI3K and mTOR kinases are multiple and complex, including common substrates, negative feedback loops, or direct activation mechanisms. First-generation allosteric mTOR inhibitors (eg, rapamycin) are mainly active on mTORC1 and mostly display cytostatic anti-tumor activity. Recently, second-generation catalytic mTOR inhibitors targeting both mTOR complexes 1 and 2 have been developed. Some of them also inhibit class IA PI3K. Here, we highlight recent data generated with these new inhibitors against cancer cells and their potential as anti-cancer drugs.

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.

Abstract B162: Inhibition of the p110α isoform of PI 3-kinase prevents PIK3CA H1047R mutant tumor growth but is associated with impaired glucose metabolism.

Abstract Signaling through the phosphoinositide 3-kinase (PI3K) pathway promotes cell survival and proliferation, but is also involved in the regulation of glucose metabolism. The PI3K pathway is frequently deregulated in cancer through amplification or mutational activation of the PIK3CA oncogene or deletion of the PTEN tumor suppressor gene. Therefore, PI3K has become a promising target for anticancer drug development, with several drugs that target multiple PI3K isoforms and mTOR (mammalian target of rapamycin) currently under clinical development. However, although class Ia PI3K oncogenic mutants have only been identified for the PIK3CA-encoded p110α isoform, there is a lack of drugs that have been developed to specifically target p110α and to identify the impact of p110α inhibition on tumor growth and glucose metabolism. In this study, we have investigated small molecule inhibitors of p110α that specifically target cells harboring the PIK3CA H1047R mutation. The selectivity of the inhibitors was confirmed by their potency against different PI3K isoforms and cellular activity was determined by their effect on pAkt expression. Tumor xenograft models with PIK3CA, PIK3CA/KRAS or PTEN mutations were used to evaluate the effect of p110α inhibition on tumor growth, while the role of p110α in glucose metabolism was investigated by tolerance to insulin, pyruvate and glucose in mice treated with PI3K inhibitors. Inhibition of p110α alone knocked down pAkt expression, with the greatest effect observed in cell lines that harbored the PIK3CA H1047R mutation and were wild-type for KRAS. A similar pattern emerged in human tumor xenograft models, where inhibition of p110 resulted in greater antitumor efficacy in SK-OV-3 (PIK3CA H1047R mutation) tumors than in HCT-116 (PIK3CA H1047R, KRAS G13D) or U87MG (PTEN deletion) tumors. In addition to delaying tumor growth, p110α inhibition also resulted in impaired glucose metabolism. The blood glucose or plasma insulin levels after administration of insulin, pyruvate or glucose were significantly elevated for up to 3 hrs in mice treated with p110α inhibitors relative to blood glucose or plasma insulin levels in control-treated mice. Similar effects on glucose metabolism were observed for pan-PI3K inhibitors, but not for selective inhibitors of other PI3K class Ia isoforms: p110α or p110α. These results suggest that selective targeting of the p110α isoform of PI3K is a viable anticancer therapeutic option, particularly for the subset of cancers harboring PIK3CA mutations. However, since p110α also appears to be necessary for regular glucose metabolism in vivo, treatment with p110α-selective PI3K inhibitors is likely to be adversely associated with hyperglycemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B162.

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein-protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity.

Enhanced Phosphoinositide 3-Kinase δ Activity Is a Frequent Event in Systemic Lupus Erythematosus That Confers Resistance to Activation-Induced T Cell Death

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease caused by the action of autoreactive T and B cells. Class I phosphoinositide-3-kinases (PI3K) are enzymes that trigger formation of 3-poly-phosphoinositides that induce cell survival. Enhanced PI3K activation is a frequent event in human cancer. Nonetheless, in a genetic model with enhanced activation of class I(A) PI3K in T cells, mice show a greater tumor index but die of a lupus-like disease. In this study, we studied the potential PI3K involvement in human SLE. The PI3K pathway was frequently activated in SLE patient PBMC and T cells (∼70% of cases), more markedly in active disease phases. We examined the mechanism for PI3K pathway activation and found enhanced activation of PI3Kδ in SLE peripheral blood T cells. The magnitude of PI3K pathway activation in patients paralleled activated/memory T cell accumulation. We examined potential tolerance mechanisms affected by increased PI3K activity; SLE patients showed reduced activation-induced cell death of activated/memory T cells. Moreover, the defective activation-induced cell death in SLE T cells was corrected after reduction of PI3Kδ activity, suggesting that PI3Kδ contributes to induction of enhanced SLE memory T cell survival. These observations point to PI3Kδ as a target of clinical interest for SLE.

Silencer of Death Domains (SODD) Inhibits Skeletal Muscle and Kidney Enriched Inositol 5-Phosphatase (SKIP) and Regulates Phosphoinositide 3-Kinase (PI3K)/Akt Signaling to the Actin Cytoskeleton

Phosphoinositide 3-kinase (PI3K) regulates cell polarity and migration by generating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) at the leading edge of migrating cells. The serine-threonine protein kinase Akt binds to PI(3,4,5)P(3), resulting in its activation. Active Akt promotes spatially regulated actin cytoskeletal remodeling and thereby directed cell migration. The inositol polyphosphate 5-phosphatases (5-ptases) degrade PI(3,4,5)P(3) to form PI(3,4)P(2), which leads to diminished Akt activation. Several 5-ptases, including SKIP and SHIP2, inhibit actin cytoskeletal reorganization by opposing PI3K/Akt signaling. In this current study, we identify a molecular co-chaperone termed silencer of death domains (SODD/BAG4) that forms a complex with several 5-ptase family members, including SKIP, SHIP1, and SHIP2. The interaction between SODD and SKIP exerts an inhibitory effect on SKIP PI(3,4,5)P(3) 5-ptase catalytic activity and consequently enhances the recruitment of PI(3,4,5)P(3)-effectors to the plasma membrane. In contrast, SODD(-/-) mouse embryonic fibroblasts exhibit reduced Akt-Ser(473) and -Thr(308) phosphorylation following EGF stimulation, associated with increased SKIP PI(3,4,5)P(3)-5-ptase activity. SODD(-/-) mouse embryonic fibroblasts exhibit decreased EGF-stimulated F-actin stress fibers, lamellipodia, and focal adhesion complexity, a phenotype that is rescued by the expression of constitutively active Akt1. Furthermore, reduced cell migration was observed in SODD(-/-) macrophages, which express the three 5-ptases shown to interact with SODD (SKIP, SHIP1, and SHIP2). Therefore, this study identifies SODD as a novel regulator of PI3K/Akt signaling to the actin cytoskeleton.

A drug targeting only p110α can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110β/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.