Class IA Phosphoinositide 3-kinase Research Articles

Regulation of Hematopoietic Stem Cell Self-Renewal and Leukemia Maintenance by the PI3K-mTORC1 Pathway

Hematopoietic stem cells (HSC) are the most well-defined stem cell population and have provided invaluable understanding of tissue-specific stem cell development and functions. Aberrant proliferation and differentiation of HSCs cause various hematologic malignancies. Studies have established that within the bulk leukemic population, there are malignant leukemic stem cells (LSCs), which can give rise to leukemia through serial transplantation. LSCs possess similar properties like HSCs, and they can both undergo self-renewal and differentiation. Similarly, HSCs and LSCs both remain in a quiescent state to prevent exhaustion due to proliferation. These processes are tightly regulated by different signaling pathways. Class IA phosphoinositide 3-kinase (PI3K)-mechanistic target of rapamycin complex (mTORC) pathway is a key pathway in regulating HSCs as well as LSCs. In this review, we focus on recent developments regarding the role of different components of the PI3K-mTORC pathway in regulating HSC and LSC functions.

Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss.

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β.

Abstract 80: PI3Kα Regulates Biomechanical Stress-induced Cytoskeletal Remodeling: A Critical Role of Gelsolin

Background: Biomechanical stress and cytoskeletal remodeling are key determinants in pressure overload-induced heart failure. Class Ia phosphoinositide 3-kinases (PI3Ks) mediate a variety of cellular activities, in response to agonist binding to cell-surface receptors, by generating the phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ) phosphoinositide lipid. Gelsolin is a Ca 2+ - and phosphoinositide-regulated actin filament severing and capping protein that is upregulated in failing human hearts and animal models of heart failure. Hypothesis: We hypothesize that PI3Kα regulates cytoskeletal remodeling through PIP 3 -mediated regulation of gelsolin. In addition, loss of gelsolin could attenuate the adverse cytoskeletal remodeling and result in increased resistance to the development of heart failure in response to pressure-overload. Methods and Results: Loss of p110α kinase activity, in two different transgenic models (PI3Kα dominant-negative (PI3KαDN) and cardiomyocyte-specific PI3Kα-null), resulted in dilated cardiomyopathy and markedly worsened cardiac dysfunction in response to transverse aortic constriction-induced pressure overload. Increased levels of mechanosensor proteins along with decreased F/G-actin ratio exhibited an uncoupling between cardiac mechanotransduction and cytoskeletal remodeling in p110α-null mice. Gelsolin activity was markedly increased in the p110α-null hearts in response to pressure-overload, whereas loss of gelsolin in PI3KαDN/gelsolin-null double mutant mice prevented the adverse cytoskeletal remodeling and preserved the cardiac function. In a murine model of chronic heart failure, loss of gelsolin prevented the pressure overload-induced cardiac dysfunction, fibrosis, and impaired cardiomyocyte contractility resulting in increased survival. Loss of gelsolin also mitigated the biomechanical stress-induced adverse cytoskeletal remodeling, via the attenuation of actin severing activity. Conclusions: We have identified a novel role of gelsolin as a mediator of adverse cytoskeletal remodeling leading to heart failure, where PI3Kα is a key regulator of gelsolin activity.

Abstract 3199: BKM120 is cytotoxic in neuroblastoma targeting the PI3K pathway

Abstract Background: Neuroblastoma is the most common extracranial solid tumor found in children, accounting for approximately 15% of cancer-related deaths. Many cellular processes have been discovered to play a role in neuroblastoma's potency, including a family of lipid kinases within the phosphoinositide 3-kinase (PI3K) signaling pathway that contribute to cell survival, proliferation, and differentiation. Targeting this pathway could unveil new treatment strategies that work to specifically treat each child's unique disease. BKM120 is a novel cancer therapeutic that targets the PI3K/Akt/mTOR signaling pathway and has recently been shown to have great potential in the clinic by acting on Class IA PI3Ks. Though Class IA PI3Ks hold multiple types, BKM120 has been shown to act preferentially on PIK3CA mutant isoforms. In this study we show the inhibitory effect of BKM120 on neuroblastoma cell lines that over-express PI3KCA, suggesting a promising role in the clinic for children with this expression profile. It has also been suggested that inhibitors of the PI3K pathway exert their inhibitory effects on cancer cells by destabilizing MYCN, a protein found over-expressed in approximately one third of neuroblastoma patients that encourages malignant progression of the disease. Methods: Neuroblastoma (NB) cell lines BE(2)-C and SMS-KCNR cells and patient lines MGT-002-13, MGT-003-08, MGT-014-11, and MGT-015-08 were used in these studies. Cell viability was measured using Calcein AM fluorescent assay at BKM120 doses 0.2uM, 0.5uM, and 1.0uM. Western blot analysis was used to measure PI3K pathway members including pAKT, p-mTOR, mTOR, and apoptosis markers including Cleaved Caspase 3, Caspase 3, PARP, and cPARP. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. IncuCyte imaging of sytox and kinetic caspase-3 reagent was used to measure apoptosis in NB cells treated with BKM120. Results: BKM120 is cytotoxic to NB cell lines with IC50's ranging from 0.9-5.5 uM. BKM120 increases protein expression levels of Cleaved Caspase 3 and cleaved PARP by inducing apoptosis and decreases MTOR, pAKT and MYCN at increasing drug doses. BKM120 decreases ATP/cell related to glycolytic metabolism activity in NB cell lines. Increased expression of MTOR and PI3KCD correlate with increased resistance to BKM120. Conclusion: This study indicates that BKM120 targets the MTOR/PI3K/AKT pathway and may play a role in MYCN driven tumors. In addition, BKM120 inhibits NB cell proliferation and induces caspase-mediated apoptosis in vitro in NB. Given the current lack of effective treatments and the high incidence of relapse and metastatic disease for patients, further assessment in clinical trial setting is needed to assess BKM120's therapeutic activity for children with NB. Citation Format: Monica M. Pomaville, Ping Zhao, Sarah DeCou, Abhinav B. Nagulapally, Jeffrey Bond, Giselle L. Saulnier Sholler. BKM120 is cytotoxic in neuroblastoma targeting the PI3K pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3199.

Abstract 129: Overexpression of P110alpha Induces Vascular Malformation in Mouse Tail, Feet and Ears

Phosphoinositide-3 kinases (PI3Ks) are a family of signal transduction enzymes involved in critical cellular functions such as cell growth, proliferation, differentiation, motility and survival. Class IA PI3K is composed of a heterodimer between a P110 catalytic subunit and a p85 regulatory subunit. Upregulation of P110alpha protein significantly propagates second messenger response accelerating Akt signaling. To augment Akt activity in macrophages, we generated mice with conditional myeloid lineage-specific (LysM-Cre) overexpression of P110alpha (M-P110α+). Peritoneal macrophages isolated from these mice expressed an additional band slightly different in size than P110alpha protein and had markedly increased phosphorylation of Akt. These mice also exhibited dramatic changes in blood cells including lower levels of white blood cells, B-cells and an increase in neutrophils; whereas the levels of T-cells and monocytes were not different compared to wild-type mice. Because of the defects of B-cell development, M-P110α+ mice had also splenomegaly with decreased levels of follicles. Unexpectedly, M-P110α+ mice at age of 3-4 weeks spontaneously developed a striking phenotype with massive enlargements of small vessels forming malformations in the extremities including tail, feet and ears. These vascular malformations grew very rapidly, and, at age 5-7 months, M-P110α+ mice developed severe hemorrhages from lesions on the tail and feet. The malformations appeared to be mainly venous by Doppler color ultrasonography, and histologically they represented overgrowth of venous and capillary cells that expressed endothelial markers including CD31/PECAM-1, von Willibrand factor and panendothelial cell antigen, MECA-32. These endothelial cells exhibited high levels of proliferation detected by Ki67 staining. In addition, the malformations were surrounded by high numbers of macrophages. In conclusion, M-P110α+ mice develop spontaneous capillary-venous malformations that represent a novel model of the human PIK3CA-related overgrowth spectrum. This mouse model provides a unique opportunity to develop novel therapeutic strategies to prevent capillary and venous malformations.

Clinical and immunologic phenotype associated with activated phosphoinositide 3-kinase δ syndrome 2: A cohort study

Activated phosphoinositide 3-kinase δ syndrome (APDS) 2 (p110δ-activating mutations causing senescent Tcells, lymphadenopathy, and immunodeficiency [PASLI]-R1), a recently described primary immunodeficiency, results from autosomal dominant mutations in PIK3R1, the gene encoding the regulatory subunit (p85α, p55α, and p50α) of class IA phosphoinositide 3-kinases. We sought to review the clinical, immunologic, and histopathologic phenotypes of APDS2 in a genetically defined international patient cohort. The medical and biological records of 36 patients with genetically diagnosed APDS2 were collected and reviewed. Mutations within splice acceptor and donor sites of exon 11 of the PIK3R1 gene lead to APDS2. Recurrent upper respiratory tract infections (100%), pneumonitis (71%), and chronic lymphoproliferation (89%, including adenopathy [75%], splenomegaly [43%], and upper respiratory tract lymphoid hyperplasia [48%]) were the most common features. Growth retardation was frequently noticed (45%). Other complications were mild neurodevelopmental delay (31%); malignant diseases (28%), most of them being B-cell lymphomas; autoimmunity (17%); bronchiectasis (18%); and chronic diarrhea (24%). Decreased serum IgA and IgG levels (87%), increased IgM levels (58%), B-cell lymphopenia (88%) associated with an increased frequency of transitional Bcells (93%), and decreased numbers of naive CD4 and naive CD8 cells but increased numbers of CD8 effector/memory Tcells were predominant immunologic features. The majority of patients (89%) received immunoglobulin replacement; 3 patients were treated with rituximab, and 6 were treated with rapamycin initiated after diagnosis of APDS2. Five patients died from APDS2-related complications. APDS2 is a combined immunodeficiency with a variable clinical phenotype. Complications are frequent, suchassevere bacterial and viral infections, lymphoproliferation, and lymphoma similar to APDS1/PASLI-CD. Immunoglobulin replacement therapy, rapamycin, and, likely in the near future, selective phosphoinositide 3-kinase δ inhibitors are possible treatment options.

ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis

Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood.Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo.In vitro activation of naive CD4+ T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4+ T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4+ T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms.Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

The class I phosphoinositide 3-kinases α and β control antiphospholipid antibodies-induced platelet activation.

Antiphospholipid syndrome (APS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies (aPL) associated with increased thrombotic risk and pregnancy morbidity. Although aPL are heterogeneous auto-antibodies, the major pathogenic target is the plasma protein β2-glycoprotein 1. The molecular mechanisms of platelet activation by aPL remain poorly understood. Here, we explored the role of the class IA phosphoinositide 3-kinase (PI3K) α and β isoforms in platelet activation by aPL. Compared to control IgG from healthy individuals, the IgG fraction isolated from patients with APS potentiates platelet aggregation induced by low dose of thrombin in vitro and increases platelet adhesion and thrombus growth on a collagen matrix under arterial shear rate through a mechanism involving glycoprotein Ib (GPIb) and Toll Like Receptor 2 (TLR-2). Using isoforms-selective pharmacological PI3K inhibitors and mice with megakaryocyte/platelet lineage-specific inactivation of class IA PI3K isoforms, we demonstrate a critical role of the PI3Kβ and PI3Kα isoforms in platelet activation induced by aPL. Our data show that aPL potentiate platelet activation through GPIbα and TLR-2 via a mechanism involving the class IA PI3Kα and β isoforms, which represent new potential therapeutic targets in the prevention or treatment of thrombotic events in patients with APS.

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

The p110α isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides (PIs). They are responsible for coordinating a diverse range of cellular functions. Class IA PI3K is a heterodimeric protein composed of a regulatory p85 and a catalytic p110 subunit. In this study, we conditionally deleted the p110α-subunit of PI3K in cone photoreceptor cells using the Cre-loxP system. Cone photoreceptors allow for color vision in bright light (daylight vision). Cone-specific deletion of p110α resulted in cone degeneration. Our studies suggest that PI3K signaling is essential for cone photoreceptor functions.

Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils.

Neutrophils play critical roles in innate immunity and host defense. However, excessive neutrophil accumulation or hyper-responsiveness of neutrophils can be detrimental to the host system. Thus, the response of neutrophils to inflammatory stimuli needs to be tightly controlled. Many cellular processes in neutrophils are mediated by localized formation of an inositol phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), at the plasma membrane. The PtdIns(3,4,5)P3 signaling pathway is negatively regulated by lipid phosphatases and inositol phosphates, which consequently play a critical role in controlling neutrophil function and would be expected to act as ideal therapeutic targets for enhancing or suppressing innate immune responses. Here, we comprehensively review current understanding about the action of lipid phosphatases and inositol phosphates in the control of neutrophil function in infection and inflammation.

Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110β, p110γ, and p110δ in pituitary gonadotropes and somatotropes.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110β, p110γ, and p110δ). Importantly, p110β and p110γ, but not p110α or p110δ, can be directly activated by the Gβγ heterodimer of Gαβγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110β, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110β only regulate basal LH secretion. However, p110β and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110β and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gβγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

TGF-β regulates the proliferation of lung adenocarcinoma cells by inhibiting PIK3R3 expression.

PIK3R3, an isoform of class IA phosphoinositide 3-kinase (PI3K), specifically interacts with cell proliferation regulators, such as retinoblastoma and proliferation cell nuclear antigen, to promote cell proliferation. However, the mechanisms behind the upstream signaling pathway of PIK3R3 remain unclear to date. This study showed that PIK3R3 expression was regulated by transforming growth factor-β (TGF-β) signaling and that PIK3R3 mediated the TGF-β-induced inhibition of lung adenocarcinoma cell proliferation. TGF-β down-regulated PIK3R3 expression in lung adenocarcinoma cells. However, this TGF-β-induced inhibition of cell proliferation can be attenuated by PIK3R3 overexpression. In addition, TGF-β can attenuate the transcriptional activity of NKX2.1, a transcription factor that binds to the promoter of PIK3R3. This result indicated that TGF-β regulated PIK3R3 expression by targeting NKX2.1. We confirmed the correlation between NKX2.1 and PIK3R3 in clinical samples. Therefore, the TGF-β/NKX2.1/PIK3R3 axis is crucial in the TGF-β-induced inhibition of cell proliferation, and the NKX2.1/PIK3R3 axis might become a target in TGF-β receptor-repressed lung adenocarcinoma.

Targeting Class IA PI3K Isoforms Selectively Impairs Cell Growth, Survival, and Migration in Glioblastoma

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancer and plays a crucial role in glioblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K isoforms as a novel anti-tumor approach in glioblastoma. Consistent expression of the PI3K catalytic isoform PI3K p110α was detected in a panel of glioblastoma patient samples. In contrast, PI3K p110β expression was only rarely detected in glioblastoma patient samples. The expression of a module comprising the epidermal growth factor receptor (EGFR)/PI3K p110α/phosphorylated ribosomal S6 protein (p-S6) was correlated with shorter patient survival. Inhibition of PI3K p110α activity impaired the anchorage-dependent growth of glioblastoma cells and induced tumor regression in vivo. Inhibition of PI3K p110α or PI3K p110β also led to impaired anchorage-independent growth, a decreased migratory capacity of glioblastoma cells, and reduced the activation of the Akt/mTOR pathway. These effects were selective, because targeting of PI3K p110δ did not result in a comparable impairment of glioblastoma tumorigenic properties. Together, our data reveal that drugs targeting PI3K p110α can reduce growth in a subset of glioblastoma tumors characterized by the expression of EGFR/PI3K p110α/p-S6.

Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.

Abstract B218: Drug-drug interaction assessment of GDC-0032, a beta-sparing inhibitor of phosphoinositide 3-kinase (PI3K), using midazolam, a sensitive CYP3A4 substrate.

Abstract Background: GDC-0032 is an orally-bioavailable, potent, and selective inhibitor of Class IA PI3K alpha, delta, gamma, and p110 beta isoforms, with 30 fold less inhibition of the p110 beta isoform. GDC-0032 has shown promising preliminary clinical activity in PIK3CA-mutant cancers and is being explored in solid tumors and in combination with endocrine therapies in breast cancer. In vitro, GDC 0032 was not a potent inhibitor of CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4 (IC50 > 30 µM) but displayed weak time-dependent inhibition of CYP3A4, with KI and kinact values of 77 µM and 0.030 min-1, respectively. In addition, GDC-0032 induced CYP3A4, but not CYP1A2 or CYP2B6 in human hepatocytes. Based on these data, the potential for GDC-0032 to impact the pharmacokinetics (PK) of midazolam (MDZ), a sensitive in vivo marker of CYP3A4 enzymatic activity, was assessed in a Phase I trial in patients with cancer. Methods: Twelve patients with advanced or metastatic solid tumors were enrolled. All patients received 5 mg MDZ on Day 1 (D1), followed by continuous daily dosing of GDC-0032 (9 mg) starting on D2. A second MDZ dose was given on D16. Predose, 0.5, 1, 1.5, 2, 3, 4, 8, and 24 hr post-dose plasma samples were collected on D1 and D16. Maximum concentration (Cmax) and area under the concentration-time curve (AUC0-24) of MDZ were estimated by non-compartmental methods using WinNonlin (version 5.2.1). MDZ plasma Cmax and AUC0-24 without GDC-0032 (D1) and after 15 daily doses of GDC-0032 (D16) were compared. The geometric mean ratios (GMRs) of Cmax and AUC0-24 and the corresponding 90% CIs were estimated (WinNonlin version 5.2.1). Physiologically-Based Pharmacokinetic (PBPK) modeling was used to prospectively predict the DDI potential of GDC-0032 (Simcyp version 11.0 and Gastroplus version 8.0). Results: No trend in individual Cmax or AUC0-24 of MDZ was observed post-GDC-0032 treatment relative to pre-treatment. The change in MDZ exposure (Cmax and AUC0-24) before and after GDC-0032 treatment was less than 2-fold for all patients (median: 1.1 fold; range: 0.4-1.9). The GMRs (90% CIs) for MDZ+GDC-0032 relative to MDZ alone for Cmax and AUC0-24 were 0.98 (0.70-1.37) and 1.04 (0.68-1.60), respectively. Prospective PBPK modeling accurately predicted a lack of marked DDI between GDC-0032 and MDZ (1-1.4 fold change). Conclusions: Despite in vitro results suggesting that GDC-0032 may impact the PK of concomitant medications which are metabolized by CYP3A4, co-administration with MDZ did not change the PK of MDZ in patients with cancer. PBPK modeling predicted little to no impact of GDC-0032 on the PK of MDZ, consistent with the in vivo observation. These results indicate that GDC-0032 may be given concomitantly with CYP substrates with minimal risk of a PK drug-drug interaction. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B218. Citation Format: Richard A. Graham, Sravanthi Cheeti, Laurent Salphati, Tong Lu, Carla Kurkjian, Daniel Von Hoff, Jasgit Sachdev, Dejan Juric, Jose Baselga, Hema Parmer, Jerry Hsu, Jeffrey Infante. Drug-drug interaction assessment of GDC-0032, a beta-sparing inhibitor of phosphoinositide 3-kinase (PI3K), using midazolam, a sensitive CYP3A4 substrate. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B218.

Signaling via Class IA Phosphoinositide 3-Kinases (PI3K) in Human, Breast-Derived Cell Lines

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN−/− cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN−/− MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.

PI3K Stimulates DNA Synthesis and Cell-Cycle Progression via Its p55PIK Regulatory Subunit Interaction with PCNA

Previously, we have shown that p55PIK, an isoform of class I(A) phosphoinositide 3-kinase (PI3K), specifically interacts with important cell-cycle regulators, such as retinoblastoma (Rb), to promote cell-cycle progression. Here, we used the glutathione S-transferase pull-down assay to identify other p55PIK-interacting proteins besides Rb in a Rb-deficient cell line and found that p55PIK interacted with proliferation cell nuclear antigen (PCNA), which plays a key role in coordinating both initiation of the leading strand DNA replication and discontinuous lagging strand synthesis. Overexpression of p55PIK increased, and knockdown decreased, DNA synthesis and DNA replication by modulating the binding of DNA polymerase δ (Polδ) to PCNA. Moreover, a cell-permeable peptide containing the N-terminal-binding domain of p55PIK (TAT-N24) disrupted the p55PIK-PCNA interaction in cancer cells, and also inhibited the DNA synthesis and tumor growth in cell culture and in vivo. Altogether, our results show that the p55PIK-PCNA interaction is important in regulating DNA synthesis and contributes to tumorigenesis. Furthermore, the p55PIK-PCNA interaction provides a potential new target for anticancer drug development.

Specific inhibition of PI3K p110δ inhibits CSF‐1‐induced macrophage spreading and invasive capacity

Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3'-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M-/-.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis.

Class I Phosphoinositide-3-Kinases and Src Kinases Play a Nonredundant Role in Regulation of Adhesion-Independent and -Dependent Neutrophil Reactive Oxygen Species Generation

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30-40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.