Phosphoinositide 3-kinase Alpha Research Articles

Targeting the PI3K/AKT/mTOR pathway with MLN0128 (mTORC1/2 inh) and MLN1117 (PI3K alpha inh) in bladder cancer: Rational for its testing in clinical trials.

369 Background: PI3K/AKT/mTOR pathway is a promising target for cancer treatment being commonly deregulated in human bladder tumors and resulting in the promotion of tumor cell growth, survival, and resistance to chemotherapy. The aim of this study is to characterize the effects of MLN0128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, and MLN1117, an orally bioavailable inhibitor of the class I phosphoinositide 3-kinase (PI3K) alpha isoform that may be more efficacious and less toxic than pan-PI3K inhibitors as bladder cancer therapies. Methods: We evaluated the effects of MLN0128 and MLN1117 both as single agents and in combination with each other or with a SOC chemotherapy agent (paclitaxel). The effects of the agents alone or in combination were analysed in a panel of six bladder cancer cell lines and in tumor xenografts. These models were selected based on specific genomic alterations that could be considered as potential therapeutic targets (PIK3CA and TSC mutations). Molecular effects of both agents and the combinations on cell-cycle, apoptosis, autophagy and on cell viability were tested in the bladder cancer cell lines. The in vivo effects on tumor growth inhibition were also assessed. Results: Both MLN0128 and MLN1117 inhibit the PI3K/AKT/mTOR pathway and reduce cell proliferation in bladder cancer cell lines with diverse genetic backgrounds. Combination of MLN0128 + MLN1117 produced synergistic antiproliferative effects in cell lines and improved the effect of each drug alone in vitro and in vivo, with no signs of toxicity in these models. Similar effects were observed with the combination of paclitaxel + MLN0128. Conclusions: Our results show that MLN0128 and MLN1117 are promising investigational agents that might be of value for bladder cancer patients. Further investigation as novel anti-cancer agents alone or in combination with chemotherapy in clinical trials in humans is warranted.

Imidazo[1,2-a]pyrazine inhibitors of phosphoinositide 3-kinase alpha (PI3Kα): 3D-QSAR analysis utilizing the Hybrid Monte Carlo algorithm to refine receptor-ligand complexes for molecular alignment

Phosphoinositide 3-kinase alpha (PI3Kα) is a lipid kinase involved in several cellular functions such as cell growth, proliferation, differentiation and survival, and its anomalous regulation leads to cancerous conditions. PI3Kα inhibition completely blocks the cancer signalling pathway, hence it can be explored as an important therapeutic target for cancer treatment. In the present study, docking analysis of 49 selective imidazo[1,2-a]pyrazine inhibitors of PI3Kα was carried out using the QM-Polarized ligand docking (QPLD) program of the Schrödinger software, followed by the refinement of receptor–ligand conformations using the Hybrid Monte Carlo algorithm in the Liaison program, and alignment of refined conformations of inhibitors was utilized for the development of an atom-based 3D-QSAR model in the PHASE program. Among the five generated models, the best model was selected corresponding to PLS factor 2, displaying the highest value of Q2test (0.650). The selected model also displayed high values of r2train (0.917), F-value (166.5) and Pearson-r (0.877) and a low value of SD (0.265). The contour plots generated for the selected 3D-QSAR model were correlated with the results of docking simulations. Finally, this combined information generated from 3D-QSAR and docking analysis was used to design new congeners.

Selective Inhibition Of The PI3K-Alpha Isoform Blocks Myeloma Cell Growth and Survival

IntroductionBoth, the phosphoinositide-3kinase (PI3K)/AKT pathway as well as its nutrient-dependent downstream target, the mToR (mammalian target of rapamycin) kinase, are essential for the growth and survival of malignant plasma cells. PI3K exists in four isotypes (α - δ), the role of each of these isotypes in multiple myeloma (MM), however, is not defined. Therefore, we evaluated systematically the in vitroanti-myeloma activity of NVP-BYL719 (Novartis), a newly developed inhibitor highly selective for the PI3K α isoform, and compared it with the activity of other inhibitors targeting the PI3K/AKT/mToR signaling network at different sites. Since inhibition of mToRC1 may lead to PI3K activation by a feedback loop, we looked also at combinations of NVP-BYL719 and rapalogs. Materials and MethodsAn MTS based growth assay was used to measure growth inhibition of NVP-BYL719 in comparison to different inhibitors (the pan-PI3K inhibitors Ly294002 and NVP-BKM120, the dual PI3K/mToR inhibitor NVP-BEZ235, and the allosteric mToR inhibitors rapamycin and everolimus) on six human malignant plasma cell lines and two non-myeloma cell lines, K562 and Raji. Western blot analysis was used to confirm inhibitor activity and specificity. In addition, combinations of PI3K and mToR inhibitors were tested and antagonistic/synergistic activity was calculated with the CalcuSynTM(Biosoft) software. ResultsThe selective PI3K alpha inhibitor NVP-BYL719 inhibited myeloma cell growth at a dose level (IC50 < 10 µM in 5/6 cell lines), where non-MM cell lines were not affected. Only cell line U266 was showed moderate sensitivity. The levels of the PI3K isoforms αwere comparable in all tested MM cell , as shown by Western blot. NVP-BYL719 blocked phosphorylation of AKT and of protein S6, an mToR downstream target. Of note, whenever rapamycin was combined with a PI3K inhibitor including NVP-BYL719, synergistic growth inhibition was seen, even in combination with the dual PI3K/mToR inhibitor NVP-BEZ235. ConclusionsHere, the PI3Kα isoform is reported as being essential for the growth of myeloma cells. This provides the rationale for the therapeutic exploration of such isoform specific PI3K inhibitors in MM allowing dose intensification with less toxicity. Allosteric mToR inhibitors, already active in early clinical trials in MM patients may provide interesting combination partners for these PI3K-inhibitors. Disclosures:Guenther:Novartis: Consultancy. Gramatzki:Novartis: Consultancy.

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 &amp;gt; 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.

Corrigendum to “Structure-based virtual screening approach to the discovery of phosphoinositide 3-kinase alpha inhibitors” [Bioorg. Med. Chem. Lett. 21 (2011) 2021–2024

Corrigendum to “Structure-based virtual screening approach to the discovery of phosphoinositide 3-kinase alpha inhibitors” [Bioorg. Med. Chem. Lett. 21 (2011) 2021–2024

Structure-based virtual screening approach to the discovery of phosphoinositide 3-kinase alpha inhibitors

Phosphoinositide 3-kinase alpha (PI3Kα) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. Herein we report a successful application of the structure-based virtual screening to identify the novel inhibitors of PI3Kα. These inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 40 μM. Therefore, they deserve a consideration for further development by structure-activity relationship (SAR) studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of PI3Kα are addressed in detail.

Negative Inotropy by Angiotensin II Is Mediated via Phosphoinositide 3-Kinase Alpha–Protein Kinase C-Coupled Signaling Pathway

Elevated plasma levels of angiotensin II have been identified as a key determinant of cardiac hypertrophy and congestive heart failure. Angiotensin II signaling mediates its biological actions in part by altering intracellular sarcoplasmic reticulum/endoplasmic reticulum calcium stores and voltage-dependent L-type Ca2+ channels.1 Calcium concentration is directly involved in cardiac excitation–contraction coupling and plays a central role in intracellular signaling pathways. Because calcium is the physiological activator of the contractile proteins, chronic elevations of cardiac angiotensin II-altered calcium handling have been postulated to underlie the calcium overload and contractile dysfunction after myocardial infarction.2 Kamp and Hell3 have suggested involvement of protein kinase A (PKA) and protein kinase C (PKC) pathways in the modulation of intracellular calcium stores and contractility. This is consistent with the reported ability of angiotensin II to promote cardiac hypertrophy via the phosphoinositide 3-kinase (PI3K)/Akt-dependent pathway.4,5 In this regard, PI3K transduces extracellular signals into cellular function by regulating activity of several genes downstream of angiotensin II type 1 receptor (AT1R).6 Based on sequence homology and substrate preference, 8 members of the PI3K family have been identified. The most widely studied isoforms of the PI3K family include PI3Kα and PI3Kγ, which regulate a variety of biological actions, including L-type calcium channel activity7 and genes associated with cell growth and survival.5 Although chronic angiotensin II stimulation reportedly leads to cardiac remodeling,8 metabolic alterations,9 cardiac fibrosis,10 and hypertrophy,5 little is known of the acute phase effects of angiotensin II on cardiomyocyte contractility. By definition, inotropy relates to alterations in contractile force generation independent of sarcomere length. It is mainly dictated by changes in activator calcium …

Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85α–p110α fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors

Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kα pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85α–p110α fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kα from which X-ray diffracting crystals were grown with inhibitors bound in the active site.

Abstract B140: In vitro sensitivity profiling identifies selectively responsive tumor types and molecular subtypes to the PI3K inhibitor GSK2126458

Abstract The phosphoinositide 3-kinase (PI3K) signalling pathway is activated in a broad spectrum of human cancers. Activation of this pathway often occurs indirectly by somatic aberrations of receptor tyrosine kinases, or directly by mutations in PI3K genes, such as PIK3CA. GSK2126458 is a novel, orally administered inhibitor of wild type phosphoinositide 3-kinase alpha (PI3K) and the common activation mutants of p110 found in human cancers that has recently entered Phase I clinical trials. In order to determine histological and genetic profiles of patients that are more likely to gain clinical benefit from GSK2126458, the compound was tested in a 3-day continuous exposure proliferation assay on a panel of 198 genetically characterized human cancer cell lines derived from a diversity of tissue types. Cells originating from bladder, breast, and ovarian cancers are most responsive based on growth inhibition metrics (gIC50's). Breast cancer cell lines harboring activating mutations of PIK3CA are associated with increased levels of response (p = 0.027); an observation which was validated in an independent cohort of 53 breast cell lines (p = 0.021). While colon cancers exhibit an intermediate level of sensitivity compared to other tumor types, specific exon 5 G12/13 hotspot mutations of the KRAS gene are predictive of decreased cellular response (p = 0.025). Additionally, a comparison of these cell line proliferation data to a similarly-assayed panel of 19 compounds of diverse target class shows that while the overall pattern of cellular response to GSK2126458 resembles other inhibitors or the PI3K/AKT/mTOR pathway, distinctions emerge that correlate with pathway targeting strategy. For example, while breast cancers are preferentially responsive to GSK2126458 and other inhibitors of this pathway, cell lines harboring PTEN mutations are less responsive to GSK2126458 than to compounds directly targeting AKT. Collectively these data suggest a unique profile for GSK2126458 among compounds that inhibit the PI3K/AKT/mTOR pathway. Positive predictors of response, such as PIK3CA mutations in breast cancer, may identify patients who are most likely to benefit from GSK2126458. The use of this predictor to select patients has the potential to reduce the size of a clinical registration trial for GSK2126458 by approximately 25%, thereby limiting cost and accelerating the development of this compound. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B140.

Synthetic lethality when combining phosphoinositide 3-kinase alpha and beta catalytic subunit-directed RNAi and estrogen deprivation for estrogen receptor positive breast cancer: implications for clinical trial design with pharmacological inhibitors.

Abstract Abstract #3063 Background: Endocrine therapy with estrogen deprivation (ED) induces cell cycle arrest in sensitive estrogen receptor positive (ER+) breast cancers, but typically not apoptosis, thus relapse is common despite ED due to residual surviving tumor cells. The phosphoinositide 3-kinase (PI3K) pathway, which promotes cell survival, is hyperactivated in ER+ breast cancer due to mutations of genes in the PI3K signaling cascade, most commonly PIK3CA. We therefore investigated the role of p110α and p110β, the gene products of PIK3CA and PIK3CB, in promoting cell survival in ER+ breast cancer.&amp;#x2028; Materials and Methods: RNA interference (RNAi) was used to selectively inhibit p110α and p110β expression in the ER+, PIK3CA mutant MCF-7 and T47D cell lines; the ER+, PIK3CA wild-type but PIK3CB gene amplified HCC712 cell line; and the ER-, PIK3CA and PIK3CB wild-type MDA-MB-231 cell line. Western blotting was used to confirm p110α and p110β knockdown efficiencies and to determine the effects of specific knockdowns on PI3K pathway signaling. Cell line growth was measured using a resazurin reduction assay. Apoptosis was quantified using TUNEL/Hoechst staining. Gene copy number for PIK3CB was assessed by FISH in cell lines and tumor samples.&amp;#x2028; Results: PIK3CA RNAi inhibited growth and activated apoptosis in all tested ER+ cell lines under ED, whereas the addition of estrogen reversed PIK3CA RNAi-induced apoptosis. PIK3CB RNAi inhibited proliferation and promoted apoptosis in the absence of estrogen only in the PIK3CB-gene amplified HCC712 cell line. In combination, PIK3CA and PIK3CB gene knockdowns produced a synergistic apoptotic effect on T47D and HCC712 cell lines, but not in MCF-7 cells which contain low levels of p110β protein. In contrast PIK3CA and PIK3CB RNAi did not promote apoptosis in the ER- MDA-MB-231 cell line. Finally, since PIK3CB gene amplification has not been described previously we conducted a screen by FISH and array CGH and detected low-level PIK3CB copy number gain or amplification in ∼5% of clinical samples from a series of breast cancer patients representing diverse clinical phenotypes (ER+, ER-, ERBB2+).&amp;#x2028; Conclusions: Inhibition of the PI3K pathway through suppression of PI3K catalytic subunit expression produces a synthetic lethal effect in the absence of estrogen specifically in ER+ breast cancer cells. These data establish a preclinical rationale for combining endocrine therapy with PI3K pharmacological inhibitors in ER+ breast cancer, but suggest that both PIK3CA and PIK3CB are therapeutic targets and gene mutation or amplification in both catalytic subunits may have to be taken into account when identiying a sensitive population for PI3 kinase-directed therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3063.

Mutations of the Catalytic Subunit of Class IA PI3K Are Constitutively Active and Confer Factor-Independent Growth to Early Hematopoietic Cells.

Background: Activating mutations of the catalytic subunit of class IA phosphoinositide 3-kinase alpha (PIK3CA) are clustered in small hot-spot regions of the PIK3CA gene, including exon 9 within the helical domain and exon 11 within the kinase domain. They have been linked to several human neoplasias, including colorectal, breast and hepatocellular cancers. In acute leukemias, PIK3CA mutations have not been investigated in larger chorts and so far only been observed in a few patients. Since the PI3K/Akt/GSK3beta pathway is an important signaling cascade of receptor tyrosine kinases (e.g. Flt3R, Kit) which are frequently activated in acute leukemias we investigated the functional activity of PIK3CA mutants.Materials and methods: We transfected early hematopoietic cells (Ba/F3 cell line) with PI3KCA exon 9 and 11 mutations and investigated the cells in an in vitro factor-independent growth assay and pharmacologic inhibition experiments.Results: We demonstrate that mutations in the helical or kinase domain of PIK3CA lead to the constitutive activation of PI3Kalpha in Ba/F3 cells, inducing factor-independent growth of the IL3-dependent cells. The frequency of IL3-independent Ba/F3 cells after tranfection with exon 9 and 11 PIK3CA mutants was equivalent to the frequency confered by PIK3CA mutants containing the membrane localization signal of either src or ras. Proliferation and survival of the cells were inhibited by the PI3K inhibitors LY294002 and Quercitin or an inhibitor of the PI3K downstream target Akt. Inhibition occurred in a dose- and time-dependent manner and could be reverted by addition of IL-3. One of the major targets of PI3K/Akt signaling is GSK3beta which becomes inactivated after Akt-mediated phosphorylation. By using a GSK3beta-specific inhibitor or LiCl we could show that the inactivation of GSK3beta alone did not result in factor-independent growth of Ba/F3 cells. However, GSK3beta inhibition led to a delay in the induction of cell death after IL3-withdrawal.Conclusion: Activating mutations of PIK3CA, associated with several human neoplasias and acute leukemia are functionally active in hematopoietic cells, confer factor independency and respond to PI3K/Akt inhibition.