Potent Phosphatidylinositol 3-kinase Inhibitor Research Articles

Rational drug design from phosphatidylinositol 3-kinase-α inhibitors through molecular docking and 3D-QSAR methodologies for cancer immunotherapy

Dysregulation or aberrant activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is commonly observed in various cancers and is associated with tumor growth, metastasis, and resistance to therapy. Targeting PI3K-α with appropriate inhibitors can disrupt this pathway, hindering cancer progression, and potentially enhancing the immune system’s ability to recognize and eliminate cancer cells. In this study, we aimed to design a novel and potent inhibitor of PI3K-α for cancer immunotherapy using rational drug design techniques, including virtual screening, molecular docking, and 3D-QSAR. We obtained the human PI3K-α protein (6PYS) complexed with (3S)-3-benzyl-3-methyl-5-[5-(2-methylpyrimidin-5-yl)pyrazolo[1,5-a]pyrimidin-3-yl]-1,3-dihydro-2H-indol-2-one (PJ5) from the RCSB Protein Data Bank. Virtual screening of ligands, integrated with predictive computational molecular docking and 3D-field-based-QSAR, was implemented using appropriate Schrödinger Maestro modules. Rational drug design was also carried out, and its clinical relevance was validated across several ADMET descriptors. Docking results suggested that a hybrid of sulfonamide and pyridine-based heterocyclic compounds, functionalized with potent moieties derived from alkaloids, exhibited adequate synergistic biological effects capable of enhancing sufficient biological activity against PI3K-α. A field-based 3D-QSAR model was built on four partial least squares factors, and five statistical metrics were employed to validate the model. The newly designed ligand from this approach, named 6’-amino-5’-(2-fluoro-1,3-oxazol-5-yl)-N-{[3-(hydroxymethyl)oxetan-3-yl]methyl}-3-methyl-[2,3’-bipyridine]-6-sulfonamide or T85, exhibited a predicted bioactivity (pIC50) of 8.25. The predicted ADMET properties of T85 fell reasonably within the range of recommended standards, especially adhering to Lipinski’s rule of five and Jorgensen’s rule of three. In conclusion, the results of this study offer significant insights into in silico drug design using a rational approach, which could expedite the discovery and development of new drug molecules.

Wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, suppresses methamphetamine-induced stereotyped sniffing and biting, ameliorating the frequency of total stereotypy in mice

Wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, suppresses methamphetamine-induced stereotyped sniffing and biting, ameliorating the frequency of total stereotypy in mice

Pretreatment with rosavin attenuates PM2.5-induced lung injury in rats through antiferroptosis via PI3K/Akt/Nrf2 signaling pathway.

Inflammation and oxidative stress caused by fine particulate matter (PM2.5) increase the incidence and mortality rates of respiratory disorders. Rosavin is the main chemical component of Rhodiola plants, which exerts anti-oxidative and antiinflammatory effects. In this research, the potential therapeutic effect of rosavin was investigated by the PM2.5-induced lung injury rat model. Rats were instilled with PM2.5 (7.5mg/kg) suspension intratracheally, while rosavin (50 mg/kg, 100 mg/kg) was delivered by intraperitoneal injection before the PM2.5 injection. It was observed that rosavin could prevent lung injury caused by PM2.5. PM2.5 showed obvious ferroptosis-related ultrastructural alterations, which were significantly corrected by rosavin. The pretreatment with rosavin downregulated the levels of tissue iron, malondialdehyde, and 4-hydroxynonenal, and increased the levels of glutathione. The expression of nuclear factor E2-related factor 2 (Nrf2) was upregulated by rosavin, together with other ferroptosis-related proteins. RSL3, a specific ferroptosis agonist, reversed the beneficial impact of rosavin. The network pharmacology approach predicted the activation of rosavin on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. LY294002, a potent PI3K inhibitor, decreased the upregulation of Nrf2 induced by rosavin. In conclusion, rosavin prevented lung injury induced by PM2.5 stimulation and suppressed ferroptosis via upregulating PI3K/Akt/Nrf2 signaling pathway.

Targeting the Glucagon Receptor Signaling Pathway As a Novel Strategy to Counteract PI3K Inhibitor Induced Hyperglycemia While Sustaining Tumor PI3K Inhibition

Pathologic activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is an oncogenic driver for many malignancies, including lymphomas1. Although validated as a therapeutic oncologic target, the PI3K signaling pathway is also implicated in normal glucose homeostasis. Specifically, since the PI3K subunit p110α is chiefly responsible for downstream insulin receptor (INSR) signaling, PI3K signaling inhibition that includes p110α leads to severe hyperglycemia. Both compensatory endogenous insulin release as well as treatment of hyperglycemia with exogenous insulin/insulin mimetics activate INSR-associated PI3K signaling, ultimately limiting blockade of tumor associated PI3K signaling2. Aggressive lymphomas driven by pathologic PI3K signaling respond to combined PI3Kα and δ/γ inhibition, but this is likely submaximal due to endogenous insulin feedback, and more intense dosing is difficult to achieve safely due to severe hyperglycemia3. Therefore, a novel strategy to maintain blockade of tumor associated PI3K signaling while reducing hyperglycemia is needed. We hypothesized that inhibition of glucagon receptor (GCGR) signaling, a pathway that does not depend on PI3K, may normalize PI3K inhibitor induced hyperglycemia without disrupting antitumor PI3K blockade. GCGR blockade with a GCGR specific monoclonal antibody (REMD-477, a human anti-GCGR antibody, or REMD2.59c, a murine equivalent) may allow for the safer use of more potent PI3K inhibitors or more intensive dosing schedules in the treatment of aggressive lymphomas. The effect of GCGR blockade on PI3K inhibitor induced hyperglycemia was first evaluated in a non-tumor bearing mouse model using REMD2.59c. The PI3K inhibitor copanlisib was administered by tail vein injection to CB17 SCID mice in the control group every other day at the reported MTD of 15 mg/kg. REMD2.59c was administered by intraperitoneal injection to the treatment group intermittently on the day prior to copanlisib injection. Blood glucose levels were measured 2 hours after copanlisib injection. Significant hyperglycemia was observed after copanlisib treatment, and this was corrected by REMD2.59c pretreatment, confirming the effectiveness of GCGR blockade (Figure A-B). Having demonstrated that GCGR blockade controls hyperglycemia caused by PI3K inhibition with copanlisib in a mouse model, we next treated a 54 year old non-diabetic woman with relapsed and refractory peripheral T cell lymphoma (PTCL) on an IRB-approved clinical pilot study of copanlisib in combination with REMD-477. Following copanlisib 60mg IV alone, glucose increased from a baseline of 100mg/dl to a maximum of 592 mg/dl at 5 hours. For the following weekly doses of copanlisib, she was pre-treated with 70mg REMD-477 SQ, and copanlisib induced hyperglycemia was significantly ameliorated (Figure C). The patient tolerated treatment with REMD-477 well without significant episodes of hypoglycemia. With improved glycemic control on REMD-477, our patient did not require dose reductions or treatment delays, and experienced clinical improvement in lymphadenopathy on copanlisib. She had previously progressed through 6 lines of therapy, including duvelisib, a selective PI3K δ/γ inhibitor which necessitated dose reduction due to gastrointestinal toxicity. Our study of the GCGR mAb REMD-477 as a novel strategy to counteract PI3K inhibitor induced hyperglycemia and insulin feedback is an important advance that may allow for more effective and safer use of potent PI3K inhibitors in the treatment of aggressive lymphomas.

Discovery and Optimization of 2-Amino-4-methylquinazoline Derivatives as Highly Potent Phosphatidylinositol 3-Kinase Inhibitors for Cancer Treatment.

Increased phosphatidylinositol 3-kinase (PI3K) signaling is among the most common alterations in cancer, spurring intensive efforts to develop new cancer therapeutics that target this pathway. In this work, we discovered a series of novel 2-amino-4-methylquinazoline derivatives through a hybridization and subsequent scaffold hopping approach that were highly potent class I PI3K inhibitors. Lead optimization resulted in several promising compounds (e.g., 19, 20, 37, and 43) with nanomolar PI3K potencies, prominent antiproliferative activities, favorable PK profiles, and robust in vivo antitumor efficacies. More interestingly, compared with 19 and 20, 37 and 43 demonstrated improved brain penetration and in vivo efficacy in an orthotopic glioblastoma xenograft model. Furthermore, preliminary safety assessments including hERG channel inhibition, AMES, CYP450 inhibition, and single-dose toxicity were performed to characterize their toxicological properties.

Icariin, but Not Genistein, Exerts Osteogenic and Anti-apoptotic Effects in Osteoblastic Cells by Selective Activation of Non-genomic ERα Signaling

Genistein and icariin are flavonoid compounds that exhibit estrogen-like properties in inducing bone formation and reducing bone loss associated with estrogen deficiency in both preclinical and clinical studies. However, the mechanisms that are involved in mediating their estrogenic actions in bone cells are far from clear. The present study aimed to study the signaling pathways that mediate the estrogenic actions of genistein and icariin in osteoblastic cells. The effects of genistein and icariin on the activation of estrogen receptor (ER) and the downstream mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in murine osteoblastic MC3T3-E1 cells and rat osteoblastic UMR-106 cells were studied. As expected, genistein displayed higher binding affinity toward ERβ than ERα and significantly induced estrogen response element (ERE)-dependent transcription in UMR-106 cells in a dose-dependent manner. In contrast, icariin failed to bind to ERα or ERβ and did not induce ERE-dependent transcription in UMR-106 cells at 10-10 to 10-7 M. The effects of genistein (10 nM) and icariin (0.1 μM) on cell proliferation and differentiation in osteoblastic UMR-106 cells were abolished in the presence of ER antagonist ICI 182,780 (1 μM), MAPK inhibitor U0126 (10 μM), and PI3K inhibitor LY294002 (10 μM). Genistein at 10 nM rapidly induced ERK1/2 phosphorylation at 5–10 min in UMR-106 cells and the phosphorylation of ERα at both Ser118 and Ser167 in both MC3T3-E1 and transfected UMR-106 cells whereas icariin at 0.1 μM rapidly activated both ERK1/2 and Akt phosphorylation in UMR-106 cells and subsequent ERα phosphorylation at both Ser118 and Ser167 in MC3T3-E1 and transfected UMR-106 cells. Confocal imaging studies confirmed that the phosphorylation of ERα at Ser 118 and Ser 167 by genistein and icariin in MC3T3-E1 cells was mediated via MAPK- and PI3K-dependent pathway, respectively. Furthermore, our studies showed that icariin exerted stronger anti-apoptotic effects than genistein and 17β-estradiol (E2) and inhibited the cleavage of downstream caspase-3 in MC3T3-E1 cells induced by a potent PI3K inhibitor, PI828 (at 2 μM). These results indicated that the mechanisms that mediate the estrogenic actions of icariin in osteoblastic cells are different from those of genistein.

Inhibition of phosphatidylinositol-3-kinase by the furanosesquiterpenoid hibiscone C

The phosphatidylinositol-3-kinase (PI3K) pathway regulates cellular metabolism and is upregulated in many cancers, making it an attractive chemotherapeutic target. Wortmannin is a potent inhibitor of PI3K; however, its potential as a chemotherapeutic is limited due to its instability, lack of selectivity, and lengthy chemical synthesis. In contrast, hibiscone C, a structurally simpler and less studied member of the furanosteroid family, has been expediently prepared by total synthesis. We demonstrate that hibiscone C competitively inhibits PI3K activity in intact cells, slows proliferation, and induces cell death. Hibiscone C may therefore serve as a productive scaffold for the development of therapeutically relevant PI3K inhibitors.

Biological characterization of SN32976, a selective inhibitor of PI3K and mTOR with preferential activity to PI3Kα, in comparison to established pan PI3K inhibitors.

Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.

Inhibition of Phosphatidylinositol-3-Kinase by Hibiscone C in T cells

Abstract Inhibitors of Phosphatidylinositol-3-Kinase (PI3K) are of significant interest due to their ability to target an intracellular pathway, which regulates cell proliferation, growth, and migration. Mutations in PI3K can lead to an upregulation of the pathway, resulting in tumorigenesis, autoimmunity, and leukemia in the immune system, while a nonfunctional PI3K can result in immunodeficiency. Wortmannin, a known and potent PI3K inhibitor, possesses many unfavorable characteristics as a chemotherapeutic and immunotherapy drug such as its insolubility in neutral buffers, limited supply, and high toxicity in vivo, which have prevented its clinical application. The small molecule inhibitor Hibiscone C, synthesized from the readily available precursors acetone and isobutylaldehyde, shares the same structural chemistry at the binding site as Wortmannin, suggesting that it shares similar biological activity. Via analysis of phosphorylation of the downstream effector molecule Akt in activated T cells, we demonstrate that Hibiscone C also can irreversibly inhibit PI3K activity due to the presence of the furanosteroid ring, to which PI3K covalently binds in each molecule. As predicted by the lack of functional groups in Hibisconce C that are present in Wortmannin, Hibiscone C was found to be less potent of an inhibitor. Despite this reduced inhibitory potency, Hibiscone C serves as a minimalist structure with which to study the requirements for PI3K binding affinity and inhibition. This reduced structure can be manipulated in order to synthesize derivatives that can maximize potency, specificity, and solubility.

Abstract P6-12-01: Phase II study of taselisib (GDC-0032) plus fulvestrant in HER2-negative, hormone receptor-positive advanced breast cancer: Analysis by PIK3CA and ESR1 mutation status from circulating tumor DNA

Abstract Background: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently dysregulated in hormone receptor (HR)-positive breast cancer (BC), with activating mutations of PIK3CA detected in ~35–45% of patients (pts). Acquired mutations in the ESR1 gene, which encodes estrogen receptor α, may be associated with resistance to aromatase inhibitor (AI) therapy. Taselisib is a potent and selective PI3K inhibitor, with greater selectivity against mutant PI3Kα isoforms than wild-type (WT) via a unique mechanism. In phase I studies, taselisib plus fulvestrant had clinical activity and manageable tolerability in pts with HR-positive BC. We report exploratory analyses of PIK3CA and ESR1 from circulating tumor DNA (ctDNA). Methods: In this phase II, open-label, single-arm study (PMT4979g; NCT01296555), pts were postmenopausal with HER2-negative, HR-positive locally advanced or metastatic BC and progression or non-response to ≥1 prior endocrine therapy in the adjuvant or metastatic setting. Pts received taselisib (6 mg capsule orally, daily) plus fulvestrant (500 mg intramuscular on Days 1 and 15 of Cycle 1, then Day 1 of each 28-day cycle) until disease progression or unacceptable toxicity. PIK3CA-mutation testing on archival tumor tissue used the cobas® PIK3CA Mutation Test. The Sysmex Inostics' BEAMing Digital PCR platform was used for ctDNA analysis of ESR1 and PIK3CA mutations (pre-dose on Cycle 1, Day 1). Primary endpoints were objective response rate (ORR) and clinical benefit rate (CBR) in all pts and those with PIK3CA mutations. ORR was confirmed complete response (cCR) and confirmed partial response (cPR). CBR was cCR, cPR, or stable disease for ≥6 months. Secondary endpoints included safety, efficacy, pharmacokinetics, and exploratory biomarker analysis. Results: 60 pts were enrolled. Median age was 61.5 years (range 31–82). In the metastatic setting, pts had received prior chemotherapy (21.7%) and prior hormonal therapy (50.0%). 86.7% of pts had received prior treatment with an AI. 45 pts had PIK3CA mutation status from archival tumor tissue and ctDNA testing; concordance was 86.7% (39/45). ctDNA analysis, vs archival tumor tissue testing, identified 4 pts and 9 pts with PIK3CA mutations from pts with WT and unknown PIK3CA mutation status, respectively. Based on ctDNA analysis (N=60), 13 pts (21.7%) had mutations in both ESR1 and PIK3CA, 21 pts (35.0%) were 'mutation not detected' (MND) for both genes, 8 (13.3%) had ESR1 mutations and PIK3CA MND, and 18 (30.0%) had ESR1 MND and PIK3CA mutations. In pts with measurable disease at baseline, confirmed responses (all partial) were: PIK3CA mutation, 38.1% (8/21); PIK3CA MND, 8.7% (2/23); all pts, 22.7% (10/44). CBRs were: PIK3CA mutation, 42.9%; PIK3CA MND, 17.4%; all pts, 29.5%. ORR and CBR from ctDNA analyses were similar to archival tumor tissue data. Conclusions: ctDNA analysis identified PIK3CA mutations in pts with previously unknown or WT mutation status from archival tumor tissue; ORR and CBR were similar to those from archival tumor tissue suggesting that PIK3CA mutation testing from ctDNA may be used as a surrogate when tissue is unavailable. 21.7% of pts had mutations in both ESR1 and PIK3CA. Citation Format: Dickler MN, Saura C, Oliveira M, Richards DA, Krop IE, Cervantes A, Stout TJ, Jin H, Savage HM, Wilson TR, Baselga J. Phase II study of taselisib (GDC-0032) plus fulvestrant in HER2-negative, hormone receptor-positive advanced breast cancer: Analysis by PIK3CA and ESR1 mutation status from circulating tumor DNA [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-01.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.

PI3Kdelta Inhibitors Increase Genomic Instability By Upregulating Aid Expression

▪Activation-induced cytidine deaminase (AID) is a B cell-specific enzyme that initiates class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes, essential mechanisms to generate different classes of antibody and antibody diversity for the antigens. At lower frequency, AID also promiscuously introduces DNA structural lesions at non-Ig loci and it is involved in the pathogenesis of B cell lymphoma. Thus, its expression is tightly controlled in B cells to limit its genotoxic effects. Phosphatidylinositol 3-kinase (PI3K) p110δ isoform acts downstream of the B-Cell Receptor (BCR) to suppress AID expression, whereas blockade of PI3K signaling enhances the expression of AID. Potent oral PI3K inhibitors such as the p110δ inhibitor idelalisib (GS-1101, CAL-101) have been recently approved for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Waldenstrom macroglobulinemia (WM), whereas dual PI3K p110δ and γ inhibitors such as duvelisib (IPI-145) showed promising results for the treatment of CLL and other hematologic malignancies.Here we investigated whether the increased of AID expression caused by idelalisib or duvelisib induced genomic instability in normal and malignant B cells.We observed that in splenic purified mouse B cells activated with IL4 and αCD40 to induce AID expression and CSR, treatment with idelalisib and duvelisib significantly increased both AID protein and mRNA levels, compared to controls. As a result, both drugs strongly promoted CSR. The regulation of AID expression was strictly dependent on PI3K p110δ activity because a selective p110γ inhibitor (AS-604850) did not have any effect. In contrast, B cells expressing a constitutively active PI3Kδ showed suppression of CSR and reduced AID expression compared to inactive PI3Kδ.To gain insights into the degree of genomic instability induced by increased AID expression upon PI3Kδ inhibition, we applied a genome-wide translocation technique we previously developed (High-Throughput Genomic Translocation Sequencing approach, HTGTS) to identify translocation partners from DNA double strand breaks (DSBs) introduced into the c-myc locus (Chiarle et al, Cell 2011). By HTGTS, we isolated thousands of translocations from activated primary mouse B cells distributed widely across the genome. Remarkably, in B cells treated with idelalisib or duvelisib, we identified a significantly higher number of translocations in known AID off-target genes as well as novel hotspots of translocations (48 for idelalisib, 50 for duvelisib). Unbiased genome-wide analysis of translocation formation revealed a consistency in the translocation patterns, with AID target hotspots localized in the TSS region and predominantly grouped within super-enhancers and regulatory clusters. HTGTS analysis performed on activated AID knock-out (AID KO) B cells showed that the vast majority of these translocations were dependent on AID. Hence, our data demonstrate that in normal B cells PI3Kδ inhibitors increase genomic instability by an AID-dependent mechanism.Finally, we observed that both idelalisib and duvelisib increased AID expression in human EBV-immortalized and lymphoma cell lines (MCL and CLL). We adapted the HTGTS technique to human cells by introducing DSBs in the human myc locus by CRISPR/Cas9 technology and sequencing genome-wide the formation of chromosomal translocations. By this approach, we demonstrated that also in human neoplastic B cells the treatment with idelalisib or duvelisib increased the formation of translocations to known AID off targets.In conclusion, we showed that idelalisib or duvelisib increase genomic instability in normal and neoplastic B cells by enhancing AID expression. Since several B cell malignancies imply treatment with these drugs for years, these effects of PI3Kδ inhibitors on the genomic stability of B cells should be carefully taken into account for therapeutic outcomes and protocol design. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-B19: BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma

Abstract Glioblastoma (GBM) is the most common glial brain tumor and is also one of the most lethal human cancers: patients have a median survival of 15 months with a five-year survival rate of only 3%. The current standard-of-care has remained the same for the last decade and consists of maximal safe surgical resection followed by radio- and chemotherapy. GBM cells are highly infiltrative, leading to invasion of normal brain tissue by tumor cells. These invasive tumor cells render GBM a surgically incurable disease and tumor recurrence is almost inevitable, with 90% of patients developing new lesions within 2-3 cm of the original site or at distant sites in the brain. Moreover, invasion may increase further during anti-angiogenic therapy with bevacizumab and no anti-invasive approaches are yet available clinically. The phosphatidyl-inositol-3 kinase (PI3K) pathway is frequently deregulated in cancer and is activated in the majority of GBM cases due to constitutive receptor tyrosine kinase activation as well as inactivating mutations/deletions of PTEN (33%) or activating PI3K mutations (17%). In addition PI3K plays a role in cell migration in some cell types and a number of small molecule PI3K inhibitors are under investigation in oncology clinical trials. BKM-120 (Buparlisib), is a CNS-penetrant selective pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for several types of solid tumor, including GBM. We initially observed that BKM-120 is a potent anti-invasive molecule in GBM cell lines and patient-derived glioma stem-like cells in vitro. The anti-migratory effects of BKM-120 were clearly distinguishable from cytostatic and cytotoxic effects that occurred at higher drug concentrations and after a longer duration of drug exposure. The blockade of migration was reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenograft model. Mechanistically we found that GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. However, BKM-120 substantially reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition may contribute to the anti-invasive effects of BKM-120. Overall, our data suggest that the CNS-penetrant PI3K inhibitor BKM-120 may be a useful anti-migratory drug for the treatment of highly invasive tumors such as glioblastoma. Citation Format: Maria Carmela Speranza, Michal O. Nowicki, Choi-Fong Cho, E. Antonio Chiocca, Sean E. Lawler. BKM-120: a phosphatidyl-inositol-3 kinase inhibitor with anti-migratory properties in glioblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B19.

Abstract 2659: Discovery of CT365, a highly potent inhibitor of PI3K and mTOR, for cancer treatment

Abstract The Phosphatidylinositol 3-kinase (PI3K) regulates essential cellular functions including cell metabolism, growth, migration, survival and angiogenesis. PI3K are divided into three subclasses on the basis of structure, regulation and lipid substrate specificity. The class I subgroup of PI3K, which includes class IA isoforms α, β, δ and class IB isoform γ, is involved primarily in the pathogenesis of human cancer. Thus, activation of the PI3K/AKT/mTOR pathway is commonly observed in human cancer and is critical for tumor progression and resistance to anti-neoplastic drugs, including cytotoxic chemotherapy and targeted drugs. Agents targeted at PI3K/mTOR signaling pathway are believed useful to treat proliferative diseases such as cancers and pulmonary fibrosis. PI3K/mTOR dual inhibitors have enhanced efficacy and a favorable safety profile. With increased understanding of immune modulation, better strategic application of rational combinations, and greater focus on patient selection, this promising class of targeted agents may soon be able to realize its potential to treat human cancers. Our clinical candidate CT365 is a potent, orally bioavailable small molecule kinase inhibitor against class I subgroup PI3Ks and mTOR. In the biochemical assays, CT365 exhibited nanomolar activities against human PI3 kinase p110α/p85α, p110β/p85α, p110δ/p85αand p120γ, with respective IC50 values of 4, 41, 2, and 6 nM. It also inhibited mTOR with an IC50 of 4 nM. CT365 demonstrated high degree of kinase selectivity when CT365 was profiled in the KinomeScan platform against a panel of 97 kinases, with only 3 kinases showing less than 10% of control binding at 1 μM. CT365 potently suppressed tumor growth in human tumor subcutaneous xenograft models with ED50s in the range of 0.8 ∼ 1.5 mg/kg. In pharmacokinetic analysis, clearance in mice, rats, dogs and cynomolgus monkeys was 0.33, 4.83, 4.50 and 2.67 mL/min/kg; and oral bioavailability in mice, rats, dogs and monkeys was 79, 95, 108 and 60%, respectively. CT365 showed no inhibition against CYP450 subtypes 3A4, 2D6, 1A2, 2C9 and 2C19 at 10 μM and no induction at 1A2, 2B6 and 3A4. The IC50 at hERG sodium channel was also determined to be greater than 30 μM. Non-GLP 28-day toxicological studies in mice and dogs indicated that CT365 has good therapeutic window. Based on the remarkable potencies of CT365 in both in vitro and in vivo studies, and desirable pharmacokinetic and toxicological profiles, CT365 was selected as a clinical candidate for the treatment of cancer. Citation Format: Ning Xi, Yanjun Wu, Tingjin Wang, Heng Wang, Zhiyong Li, Qingwei Meng, Jing Li, Zhaohe Wang, Yingjun Zhang. Discovery of CT365, a highly potent inhibitor of PI3K and mTOR, for cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2659. doi:10.1158/1538-7445.AM2015-2659

Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells.

Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/mammalian target of rapamycin and p38 MAPK mediated pathways.

A Novel PI3K Inhibitor Identified By a High Throughput Virtual Screen Displays Potent Activity Against Multiple Myeloma

The phosphatidylinositol-3-kinase (PI3K) has been developed as a promising target for the treatment of many cancers, including hematological malignancies, such as lymphoma, leukemia, and multiple myeloma (MM). Recent clinical trials have demonstrated Idelalisib, a specific PI3Kdelta inhibitor, reached a total 57% of response rate in relapsed indolent lymphoma and 72% of overall complete rate in relapsed chronic lymphocytic leukemia. MM remains fatal as a cancer of plasma cells because of lack of efficient drugs. Therefore, we tried to find novel PI3K inhibitors for MM therapy. To this end, we performed a high throughput virtual screen against 800,000 small molecule compounds using PI3Kγ as the analytic model and identified C98, a function unknown compound, as a potent inhibitor of PI3Ks. The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino residues through which C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, and STAT3. PTEN is a key negative modulator of the PI3K signaling pathway, but KIF did not show any induction of PTEN, thus further demonstrating that KIF is a selective PI3K inhibitor. Inhibition of PI3K by C98 led to myeloma cell apoptosis as shown activated caspase cascade signaling. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analysis revealed that C98 can be found in MM tissues at an average concentration up to 622.6 ng/g, suggesting that C98 can penetrate into the tumor tissues where it exerts its anti-myeloma activity. It also suggests C98 has a reliable oral activity. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development. DisclosuresNo relevant conflicts of interest to declare.

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

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

Combine MEK inhibition with PI3K/mTOR inhibition exert inhibitory tumor growth effect on KRAS and PIK3CA mutation CRC xenografts due to reduced expression of VEGF and matrix metallopeptidase-9

Although epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab are used widely to treat KRAS wild-type metastatic colorectal cancer (mCRC), patients become resistant by various mechanisms, including KRAS, BRAF, and PIK3CA mutations, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we investigated the mechanisms of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), in a KRAS and PIK3CA mutation CRC xenograft model. HCT116 (KRAS (G13D) , PIK3CA (H1047R) mutant) cells were subcutaneously injected into the nude mice. Mice were randomly assigned to treatment with vehicle, cetuximab, AZD6244, BEZ235, or AZD6244 plus BEZ235, for up to 3weeks; then, all mice were sacrificed, and tumor tissues were subjected to Western blot analysis and immunohistochemical staining. AZD6244 or BEZ235 slightly inhibit tumor growth of HCT116 xenografts, and the combination treatment markedly enhanced their antitumor effects. However, cetuximab had no effect on tumor growth. Western blot analysis and immunohistochemical staining revealed that treatment with AZD6244 or BEZ235 could significantly reduce the phosphorylation level of ERK1/2 or AKT in HCT116 tumor tissues. More interesting, the antiangiogenic effects were substantially enhanced when the agents were combined which may due to the reduced expression of VEGF and matrix metallopeptidase-9 (MMP-9) in tumor tissues. These results suggest that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in CRC harboring with KRAS and PIK3CA mutations. The mechanisms of synergistic antitumor effects may be due to antiangiogenesis.

Abstract 3341: Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer

Abstract The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is a critical survival cascade in tumor progression. Our previous study demonstrated that combinational treatment of a potent PI3K inhibitor, LY294002 (LY), and DNA methyltransferase inhibitor, 5’-Aza-deoxycytidine (DAC), compounds effectively reduce tumor formation in the immunodeficiency mouse model, suggesting that PI3K/AKT signaling suppresses tumorigenesis through the epigenetic modulation. While tumor size of the LY-treated xenografts is dramatically decreased compared to the control group, the tumor growth cannot be completely attenuated by either LY or LY/DAC treatment, indicating that there is an alternative route for tumor survival. Here, we performed a microarray analysis to identify LY-mediated expression profiling on an ERα-positive breast cancer cell line, T-47D, the cell line applied in the previous xenograft experiments. A subset of 24 LY-activated genes associated with better clinical outcome are found to be not only direct targets of PI3K/AKT but involved in estrogen (E2)/ERα signaling by ingenuity pathway analysis, suggesting the involvement of ERα signaling in PI3K/AKT-mediated tumor growth. To clarify their relationship, pharmacological experiments using an ERα antagonist, ICI 182, 780 (ICI) and LY coupled with E2 was conducted. We observed that E2 stimulation induces both AKT and ERα activation in a time-dependent manner. Either ICI or LY treatment cannot repress the activation levels of AKT or ERα, respectively. Though, either single or combined inhibitor treatment can effectively restore E2-suppressed gene expression. These findings indicate that cross-talk between PI3K/AKT and ERα signaling cascades may cooperatively regulate cell growth and associated gene expression. In addition, transcription factor motif analysis showed that signal transducer and activator of transcription 3 and 6 (STAT3/6) binding motifs are enriched with highest prediction score on the promoter regions of the examined AKT-directed targets. Inhibitors prohibit E2-mediated assembly of STAT3/6 with ERα and STAT3/6 recruitment onto the promoter regions of target loci using co-immunoprecipitation (co-IP) and chromatin IP-qPCR. Moreover, repressive histone marks, H3K27me3 and H3K9me3, are found to occupy on the same regions as STAT3/6 upon E2 treatment. DAC treatment can effectively restore E2-induced suppression of AKT-directed target genes. Further association studies are ongoing to interrogate whether STAT3/6 is part of repressive transcription complex with H3K27me3 and H3K9me3. In summary, we propose that cross-talk of ERα with PI3K/AKT signaling represents the potential function switch of ERα from genomic binding to no-genomic binding. Combined treatment of PI3K/AKT inhibitors with hormone therapy may shed a light of therapeutic strategy to patients with aggressive or metastatic breast tumors. Citation Format: Hang-Kai Hsu, Pei-Yin Hsu, Yao Wang, Zelton D. Sharp, Tim H.-M. Huang, Victor X. Jin. Cross-talk between PI3K/AKT and ERα signaling leads to STAT3/6-modulated epigenetic transcription in aggressive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2014-3341

Antitumor activity of selective MEK1/2 inhibitor AZD6244 in combination with PI3K/mTOR inhibitor BEZ235 in gefitinib-resistant NSCLC xenograft models

PurposeAlthough the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, MET amplification, and KRAS mutation, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3–kinase (PI3K) and mammalian target of rapamycin (mTOR), in gefitinib-resistant non-small cell lung carcinoma (NSCLC) models.Experimental designNCI-H1975 with EGFR-T790M mutation, NCI-H1993 with MET amplification and NCI-H460 with KRAS/PIK3CA mutation human NSCLC cells were subcutaneous injected into the athymic nude mice respectively. Mice were randomly assigned to treatment with AZD6244, BEZ235, AZD6244 plus BEZ235, or control for 3 weeks, then all mice were sacrificed and tumor tissues were subjected to western blot analyses and immunohistochemical staining.ResultsAZD6244 could inhibit the tumor growth of NCI-H1993, but slightly inhibit the tumor growth of NCI-1975 and NCI-H460. Combining AZD6244 with BEZ235 markedly enhanced their antitumor effects and without any marked adverse events. Western blot analysis and immunohistochemical staining revealed that AZD6244 alone reduced ERK1/2 phosphorylation, angiogenesis, and tumor cell proliferation. Moreover, MEK1/2 inhibition resulted in decreased AKT phosphorylation in NCI-H1993 tumor model. BEZ235 also inhibited AKT phosphorylation as well as their downstream molecules in all three tumor models. The antiangiogenic effects were substantially enhanced when the agents were combined, which may due to the reduced expression of matrix metallopeptidase-9 in tumor tissues (MMP-9).ConclusionsIn this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. Combining AZD6244 with BEZ235 enhanced their antitumor and antiangiogenic effects. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. This new therapeutic strategy may be a practical approach in the treatment of these patients.