Potent Phosphatidylinositol 3-kinase Research Articles

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.

Formononetin Improves the Survival of Random Skin Flaps Through PI3K/Akt-Mediated Nrf2 Antioxidant Defense System.

Random-pattern skin flap is widely used in plastic and reconstructive surgery. However, its clinical effect is limited by ischemia necrosis occurs at the distal part of flap. Previous studies have proved that the protective effect of formononetin was associated with its antioxidant, anti-inflammatory ability. However, further research is still needed on the effect of formononetin on flap viability. The purpose of our study was to investigate the effect of formononetin on flap survival and the underlying mechanisms. Two doses (25 mg/kg, 50 mg/kg)of formononetin were administered for seven consecutive days on flap model. Flap tissues were collected on postoperative day 7. Our results revealed that formononetin promoted skin flap viability in a dose-dependent manner. Using immunohistochemical staining and western blot, we found that formononetin significantly reduced oxidative stress and inflammation. Hematoxylin and eosin (H and E) staining, laser Doppler images and immunofluorescence staining showed the enhancement of angiogenesis after formononetin treatment. Mechanistically, we demonstrated that the antioxidation of formononetin was mediated by activation and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), while down-regulating cytoplasmic Kelch-like ECH-associated protein 1 (Keap1) expression. Co-treatment with formononetin and LY294002 (15 mg/kg), a potent Phosphatidylinositol-3-kinase (PI3K) inhibitor, which aborted nuclear Nrf2 expression and phosphorylated Akt, indicating that formononetin-mediated Nrf2 activation was related to PI3K/Akt pathway. Overall, our findings revealed that formononetin increased angiogenesis, reduced oxidative stress and inflammation, thus promoting flap survival. We highlighted the antioxidant effects of formononetin since the Nrf2 system was activated. Therefore, formononetin might be a promising candidate drug that can enhance survival of skin flaps.

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.

Involvement of Catechols in Acteoside in the Activation of Promatrix Metalloproteinase-2 and Membrane Type-1-Matrix Metalloproteinase Expression via a Phosphatidylinositol-3-Kinase Pathway in Human Dermal Fibroblasts.

Granulation tissue formation during skin wound healing requires the migration and proliferation of dermal fibroblasts in the wound site, where a subsequent remodeling of extracellular matrices (ECM) occurs. An abnormality of ECM remodeling within the healing wound leads to fibrosis and a contracted scar. To evaluate whether acteoside, a phenylethanoid glycoside isolated from the leaves of Rehmannia glutinosa LIBOSCH., exhibits wound-healing actions, we examined the effect of acteoside on the expression of matrix metalloproteinases (MMPs) in normal human dermal fibroblasts (NHDF). Acteoside dose- and time-dependently augmented the activation of the precursor of MMP-2 (proMMP-2/progelatinase A) in untreated- and interleukin-1β-treated NHDF, while the alteration of the MMP-2 gene expression was negligible. The acteoside-induced proMMP-2 activation was associated with the augmented membrane-type 1 MMP (MT1-MMP) expression in the NHDF. In addition, the proMMP-2 activation was enhanced by two aglycones in acteoside: caffeic acid and 3,4-dihydroxyphenylethanol, which consist of catechol. However, there was no change in the proMMP-2 activation in other catechol derivatives: homovanillyl alcohol- and homovanillic acid-treated NHDF, indicating that catechols in acteoside were requisite for the regulation of the MMP activation and expression in NHDF. Furthermore, the proMMP-2 activation by acteoside was selectively inhibited by LY294002, a potent phosphatidylinositol-3-kinase (PI3K) inhibitor. These results provide novel evidence that acteoside augments proMMP-2 activation along with an increase in MT1-MMP expression through a PI3K signal pathway in NHDF. Thus, acteoside is likely to be an attractive candidate that facilitates ECM remodeling in the skin wound repair process.

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.

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.

Design, Synthesis, and Biological Evaluation of Substituted Pyrimidines as Potential Phosphatidylinositol 3-Kinase (PI3K) Inhibitors.

Three series of substituted pyrimidines were designed and synthesized. All target compounds were screened for kinase inhibitory activities against PI3Kα, and most IC50 values were found within the nanomolar range. Compounds 5d and 5p displayed comparable activities relative to the positive control 5a. 5p also showed a significant isozyme selectivity (PI3Kβ/α). Furthermore, the cytotoxicities of these pyrimidines against human cancer cell lines were evaluated and the in vivo anticancer effect of 5d was also tested.

Dehydroglyasperin D Inhibits the Proliferation of HT-29 Human Colorectal Cancer Cells Through Direct Interaction With Phosphatidylinositol 3-kinase.

Background:Despite recent advances in therapy, colorectal cancer still has a grim prognosis. Although licorice has been used in East Asian traditional medicine, the molecular properties of its constituents including dehydroglyasperin D (DHGA-D) remain unknown. We sought to evaluate the inhibitory effect of DHGA-D on colorectal cancer cell proliferation and identify the primary signaling molecule targeted by DHGA-D.Methods:We evaluated anchorage-dependent and -independent cell growth in HT-29 human colorectal adenocarcinoma cells. The target protein of DHGA-D was identified by Western blot analysis with a specific antibody, and direct interaction between DHGA-D and the target protein was confirmed by kinase and pull-down assays. Cell cycle analysis by flow cytometry and further Western blot analysis was performed to identify the signaling pathway involved.Results:DHGA-D significantly suppressed anchorage-dependent and -independent HT-29 colorectal cancer cell proliferation. DHGA-D directly suppressed phosphatidylinositol 3-kinase (PI3K) activity and subsequent Akt phosphorylation and bound to the p110 subunit of PI3K. DHGA-D also significantly induced G1 cell cycle arrest, together with the suppression of glycogen synthase kinase 3β and retinoblastoma phosphorylation and cyclin D1 expression.Conclusions:DHGA-D has potent anticancer activity and targets PI3K in human colorectal adenocarcinoma HT-29 cells. To our knowledge, this is the first report to detail the molecular basis of DHGA-D in suppressing colorectal cancer cell growth.

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.

The Guareschi Pyridine Scaffold as a Valuable Platform for the Identification of Selective PI3K Inhibitors.

A novel series of 4-aryl-3-cyano-2-(3-hydroxyphenyl)-6-morpholino-pyridines have been designed as potential phosphatidylinositol-3-kinase (PI3K) inhibitors. The compounds have been synthesized using the Guareschi reaction to prepare the key 4-aryl-3-cyano-2,6-dihydroxypyridine intermediate. A different selectivity according to the nature of the aryl group has been observed. Compound 9b is a selective inhibitor against the PI3Kα isoform, maintaining a good inhibitory activity. Docking studies were also performed in order to rationalize its profile of selectivity.

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 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.

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

Lithiation of a silyl ether: formation of an ortho-Fries hydroxyketone.

A hydroxy-directed alkylation of an N,N-diethylarylamide using CIPE-assisted α-silyl carbanions (CIPE = complex-induced proximity effect) has been developed using a simple reagent combination of LDA (lithium diisopropylamide) and chlorosilane. A study of the mechanism, and the application of the procedure to an anionic Snieckus-Fries rearrangement for a highly efficient synthesis of the potent phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, are reported.