Inhibitor BEZ235 Research Articles

Endoplasmic reticulum stress could induce autophagy and apoptosis and enhance chemotherapy sensitivity in human esophageal cancer EC9706 cells by mediating PI3K/Akt/mTOR signaling pathway.

The study was designed to explore the mechanism of tunicamycin-induced endoplasmic reticulum stress in human esophageal cancer EC9706 cells and EC109 cells, as well as its effects on cell autophagy, apoptosis, and chemoresistance. Tunicamycin-induced endoplasmic reticulum stress model was established in EC9706 and EC109 cell lines. Western blotting was employed to detect the expression of endoplasmic reticulum stress iconic protein GRP78. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate the effect of different cisplatin and tunicamycin concentrations on survival rate of EC9706 cells and EC109 cells. Autophagy was monitored using monodansylcadaverin and apoptosis was detected by flow cytometry. Western blotting was used to detect the expressions of endoplasmic reticulum stress-related proteins (PERK, eIF2α, and CHOP), PI3K/Akt/mTOR signaling pathway-related proteins, autophagy-related proteins (LC3-I/LC3-II, Beclin-1, and p62), and apoptosis-related proteins (Bcl-2, Bax, and cleaved caspase-3). Tunicamycin led to increased expression of GRP78. With tunicamycin treatment, phosphorylation of PERK and eIF2α and CHOP expression increased. Meanwhile, the increase in cytolysosome was concentration and time dependent. With the increased tunicamycin concentration, there were increased expressions of Bax and cleaved caspase-3, decreased expression of Bcl-2, and lower phosphorylation of PI3K/Akt/mTOR signaling pathway-related proteins. Therefore, it can be concluded that the combination of tunicamycin and cisplatin could improve the sensitivity of EC9706 cells and EC109 cells to cisplatin; PI3K inhibitor BEZ235 could enhance cell autophagy and apoptosis and increase cell sensitivity to cisplatin.

Targeting Metabolic Reprogramming by Influenza Infection for Therapeutic Intervention

Influenza is a worldwide health and financial burden posing a significant risk to the immune-compromised, obese, diabetic, elderly, and pediatric populations. We identified increases in glucose metabolism in the lungs of pediatric patients infected with respiratory pathogens. Using quantitative mass spectrometry, we found metabolic changes occurring after influenza infection in primary human respiratory cells and validated infection-associated increases in c-Myc, glycolysis, and glutaminolysis. We confirmed these findings with a metabolic drug screen that identified the PI3K/mTOR inhibitor BEZ235 as a regulator of infectious virus production. BEZ235 treatment ablated the transient induction of c-Myc, restored PI3K/mTOR pathway homeostasis measured by 4E-BP1 and p85 phosphorylation, and reversed infection-induced changes in metabolism. Importantly, BEZ235 reduced infectious progeny but had no effect on the early stages of viral replication. BEZ235 significantly increased survival in mice, while reducing viral titer. We show metabolic reprogramming of host cells by influenza virus exposes targetsfor therapeutic intervention.

PI3K inhibitor enhances the cytotoxic response to etoposide and cisplatin in a newly established neuroendocrine cervical carcinoma cell line.

BackgroundNeuroendocrine cervical carcinoma (NECC) is a rare and aggressive subtype of cervical cancer. To date, no NECC cell-based model is available, which hinders the development of new therapeutic strategies for NECC. In this study, we derived a new NECC cell line from an ex vivo biopsy and used it to explore novel drug combination approach for NECC.ResultsThe stable HM-1 cell line displayed high expression levels of the neuroendocrine marker, synaptophysin. HM-1 cell transplantation could induce tumor growth in nude mice. As expected, the combination of etoposide and cisplatin synergistically inhibited HM-1 cell proliferation. Strikingly, when etoposide and cisplatin were combined with PI3K inhibitor BEZ235, the growth of HM-1 cells was significantly reduced. Taken together, the data implied the combination of etoposide and cisplatin with BEZ235 not only inhibited HM-1 cell proliferation but also increased cell apoptosis.Materials and MethodsA NECC tissue sample from a 75-year-old female patient was processed to derive a primary cell line annotated as HM-1. The features of HM-1 were analyzed to establish its characteristic profile. Next, HM-1 was treated with PI3K inhibitors, BKM120 and/or BEZ235, in combination with two well-known genotoxic drugs, etoposide and/or cisplatin, to evaluate which combination could serve as a more effective treatment approach. Their inhibiting effects on HM-1 were evaluated by cell viability, apoptosis, and target kinase expression.ConclusionsThe newly established NECC cell line HM-1 could serve as a cell-based model for NECC research. The synergistic drug combination of PI3K inhibitor with genotoxic drugs might become a potential new treatment strategy against NECC.

STK11 Loss Stabilizes MCL1 and Confers Targetable Drug Resistance in KRAS Mutant NSCLC

The combined KRAS/STK11 (serine‐threonine kinase 11, LKB1) mutated genotype is prevalent and predictive of poor clinical outcome in NSCLC as tumors are drug resistant. Here, we demonstrate that the bromodomain and extraterminal inhibitor (BETi) JQ1 can induce apoptosis in KRASm NSCLC by targeting the pro‐apoptotic BIM anti‐apoptotic BCL2/BCL‐XL axis, but not in KRASm/STK11m due to additional elevated MCL1. Specifically, loss of STK11 stabilizes anti‐apoptotic MCL1 protein via activation of the PI3K‐mTOR signaling axis. In NSCLC cell line models, we have demonstrated that overexpressing myristoylated‐PI3K increased MCL1 expression whereas the PI3K/mTOR inhibitor BEZ235 reduced MCL1 in a dose, time, and proteasome‐dependent, yet relatively GSK3β‐independent, manner. Furthermore, MCL1 stability also attenuates the antitumor efficacy of the direct BCL2/BCL‐XL inhibitor, ABT‐263 (Navitoclax) in KRASm/STK11m cells. However, combination of BETi or ABT‐263 with MCL1 protein reduction by siRNA or BEZ235, or direct MCL1 inhibition by the BH3 mimetic, A1210477, could sensitize resistant cells to apoptosis while no monotherapy was effective. Therefore, STK11 mutation or aberrant PI3K/mTOR pathway activations emerge as biomarkers of BET‐ or BCL2/BCL‐XL inhibitor resistance, suggesting patient selection for combined MCL1 inhibition.Support or Funding InformationThis work is supported by research Scholar grant RSG‐14‐229‐01 from the American Cancer Society and United Against Lung Cancer Legacy Program (Lung Cancer Research Foundation). This work is also supported by research fellowship from Sumitomo life social welfare services foundation (E.K), MINECO (SAF2010‐21769), ISCIII (RD12/0036/0045) and Generalitat Valenciana (ACOMP/2013/156 and ACIF/2013/239) (J.C.), and National Institutes of Health grants CA140594, CA122794, CA163896, CA166480 (K.K.W)

Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations.

Therapeutic targeting of the PI3K pathway is an active area of research in multiple cancer types, including breast and endometrial cancers. This pathway is commonly altered in cancer and plays an integral role in numerous vital cellular functions. Mutations in the PIK3CA gene, resulting in a constitutively active form of PI3K, often occur in colorectal cancer, though the population of patients who would benefit from targeting this pathway has yet to be identified. In human colorectal cancers, PIK3CA mutations most commonly occur concomitantly with loss of adenomatous polyposis coli (APC). Here, treatment strategies are investigated that target the PI3K pathway in colon cancers with mutations in APC and PIK3CA Colorectal cancer spheroids with Apc and Pik3ca mutations were generated and characterized confirming that these cultures represent the tumors from which they were derived. Pan and alpha isomer-specific PI3K inhibitors did not induce a significant treatment response, whereas the dual PI3K/mTOR inhibitors BEZ235 and LY3023414 induced a dramatic treatment response through decreased cellular proliferation and increased differentiation. The significant treatment responses were confirmed in mice with Apc and Pik3ca-mutant colon cancers as measured using endoscopy with a reduction in median lumen occlusion of 53% with BEZ235 and a 24% reduction with LY3023414 compared with an increase of 53% in controls (P < 0.001 and P = 0.03, respectively). This response was also confirmed with 18F-FDG microPET/CT imaging.Implications: Spheroid models and transgenic mice suggest that dual PI3K/mTOR inhibition is a potential treatment strategy for APC and PIK3CA-mutant colorectal cancers. Thus, further clinical studies of dual PI3K/mTOR inhibitors are warranted in colorectal cancers with these mutations. Mol Cancer Res; 15(3); 1-11. ©2016 AACR.

Abstract P3-04-18: The impact of circulating androgens on the androgen receptor in aromatase inhibitor resistant breast cancer

Abstract Aromatase Inhibitor (AI) therapy is the gold standard first line therapy for post-menopausal breast cancer. Following AI treatment the conversion of circulating androgens into estrone can be diminished by &amp;gt;99%, completely altering the tumour steroid microenvironment. Estrogens have been extensively studied in terms of the role of sex steroids as causative agents of breast cancer but interestingly, a case–control study nested within the European Prospective Investigation into Cancer and Nutrition reported that elevated levels of both serum androgens and estrogens are associated with increased breast cancer risk (E2 RR: 2.28 and androstenedione RR: 1.94). Also clinical studies have reported increases in the serum levels of androstenedione (4AD) in patients that recur on AI therapy. In order to begin to address the role of circulating androgens and the androgen receptor (AR) in AI resistant breast cancer, RNA sequencing was performed on AI resistant (MCF7-aro-LetR) cells cultured with 4AD in the presence of letrozole. Gene expression analysis was performed using the cBioPortal software found that PI3K signalling pathways were upregulated in response to 4AD treatment as well as the upregulation of pathways involved in exocytosis and hormone secretion. We found elevated levels of PI3K and AR in endocrine resistant breast cancer compared to sensitive cell lines and that our AI resistant LetR cells showed significant reduction in cell growth in response to the combined pan class PI3K inhibitor BEZ235 and anti-AR treatment. Clinically, we evaluated the potential role of PI3K signalling in AI resistance a breast cancer patient TMA (n=488) was stained immunohistochemically for phospho-AKT and AR. The protective effect conferred by high AR expression in the total patient population (p=0.02) and tamoxifen treated population (p=0.05) was lost in the AI treated population (p=0.49). For p-AKT staining, patients that were positive for p-AKT significantly associate with the more aggressive luminal B classification yet exhibit an inverse association with luminal A subtype. Kaplan Meier analysis revealed that AR is associated with a more favourable outcome in p-AKT negative patients but that this is lost in the context of elevated p-AKT. Furthermore, interrogation of the breast invasive carcinoma TCGA dataset (n=963) revealed that patients with upregulated AR mRNA and wildtype PI3K exhibited more invasive disease compared with those harbouring a PI3K mutation (p=0.02) (n=56). Further investigations into the AR interacting partners will help elucidate potential mechanisms of action and the role of androgens and AR in facilitating breast cancer tumourigenesis. Citation Format: Creevey L, Ali A, Bleach R, Hill ADK, Young L, McIlroy M. The impact of circulating androgens on the androgen receptor in aromatase inhibitor resistant breast cancer [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 P3-04-18.

PI3K/mTOR dual inhibitor BEZ235 and histone deacetylase inhibitor Trichostatin A synergistically exert anti-tumor activity in breast cancer.

Molecule-targeted therapy has achieved great progress in cancer therapy. Effective drug combinations are one way to enhance the therapeutic efficacy and combat resistance. Here, we determined the effect of the PI3K/mTOR dual inhibitor BEZ235 and the histone deacetylase inhibitor Trichostatin A (TSA) on human breast cancer. We demonstrated that the combination of BEZ235 and TSA results in significant synergistic growth inhibition of multiple breast cancer cell lines. Mechanistic studies revealed that the combined therapy induced apoptosis in a caspase-dependent manner, which might be related to the further depression of the PI3K/Akt/mTOR signalling pathway. Additionally, co-treatment with BEZ235 and TSA enhanced autophagic cell death by up-regulating the expression of LC3B-II and Beclin-1. The vivo tumour modelling studies revealed that BEZ235 combined with TSA blocked tumour growth without noticeable side effects. These data suggest that the combination of BEZ235 and TSA may be a new selective strategy, which may have significant clinical application in the treatment of breast cancer patients.

The PI3K/mTOR dual inhibitor BEZ235 suppresses proliferation and migration and reverses multidrug resistance in acute myeloid leukemia.

Aberrant activation of the PI3K/Akt/mTOR pathway contributes to the proliferation of malignant cells, and may confer resistance to chemotherapy in various malignancies, including acute myeloid leukemia (AML). Chemoresistance is the major reason for relapse in AML. RAD001 (everolimus) has been used at d1 and d7 of an induction chemotherapy regimen for AML, which has acceptable toxicity and may improve conventional chemotherapeutic treatment. Dual inhibitors of PI3K and mTOR overcome some of the intrinsic disadvantages of rapamycin and its derivatives. In this study, we evaluated the effects of BEZ235, a PI3K/mTOR dual inhibitor, on the multidrug-resistant AML cell lines HL-60/VCR and K562/ADR in vitro. BEZ235 dose-dependently inhibited the viability of HL-60/VCR and K562/ADR cells with the IC50 values of 66.69 and 71.44 nmol/L, respectively. BEZ235 (25-100 nmol/L) dose-dependently inhibited the migration of the two AML cell lines, and it also significantly sensitized the two AML cell lines to VCR and ADR. After treatment with BEZ235, the miR-1-3p levels were markedly increased in HL-60/VCR cells. Using TargetScan analysis and luciferase assays, we showed that miR-1-3p targeted BAG4, EDN1 and ABCB1, the key regulators of cell apoptosis, migration and multidrug resistance, and significantly decreased their levels in the two AML cell lines. Transfection of HL-60/VCR and K562/ADR cells with miR-1-3p-AMO to inhibit miR-1-3p could reverse the anti-proliferation effects of BEZ235. In conclusion, the PI3K/mTOR dual inhibitor BEZ235 effectively chemosensitizes AML cells via increasing miR-1-3p and subsequently down-regulating BAG4, EDN1 and ABCB1.

A Combined ULBP2 and SEMA5A Expression Signature as a Prognostic and Predictive Biomarker for Colon Cancer.

Background: Prognostic biomarkers for cancer have the power to change the course of disease if they add value beyond known prognostic factors, if they can help shape treatment protocols, and if they are reliable. The aim of this study was to identify such biomarkers for colon cancer and to understand the molecular mechanisms leading to prognostic stratifications based on these biomarkers.Methods and Findings: We used an in house R based script (SSAT) for the in silico discovery of stage-independent prognostic biomarkers using two cohorts, GSE17536 and GSE17537, that include 177 and 55 colon cancer patients, respectively. This identified 2 genes, ULBP2 and SEMA5A, which when used jointly, could distinguish patients with distinct prognosis. We validated our findings using a third cohort of 48 patients ex vivo. We find that in all cohorts, a combined ULBP2/SEMA5A classification (SU-GIB) can stratify distinct prognostic sub-groups with hazard ratios that range from 2.4 to 4.5 (p≤0.01) when overall- or cancer-specific survival is used as an end-measure, independent of confounding prognostic parameters. In addition, our preliminary analyses suggest SU-GIB is comparable to Oncotype DX colon(®) in predicting recurrence in two different cohorts (HR: 1.5-2; p≤0.02). SU-GIB has potential as a companion diagnostic for several drugs including the PI3K/mTOR inhibitor BEZ235, which are suitable for the treatment of patients within the bad prognosis group. We show that tumors from patients with worse prognosis have low EGFR autophosphorylation rates, but high caspase 7 activity, and show upregulation of pro-inflammatory cytokines that relate to a relatively mesenchymal phenotype.Conclusions: We describe two novel genes that can be used to prognosticate colon cancer and suggest approaches by which such tumors can be treated. We also describe molecular characteristics of tumors stratified by the SU-GIB signature.

Abstract A39: Expression of alternatively spliced tissue factor in pancreatic neuroendocrine tumors

Abstract Background: Pancreatic cancer remains extremely difficult to treat, resulting in an 80% 1-year mortality rate. Tissue Factor (TF) serves as a co-factor for serine protease factor VIIa and the full-length (fl)TF/VIIa complex initiates blood coagulation. Alternatively spliced isoform of TF (asTF) occurs naturally, is minimally coagulant and, in contrast to flTF, dispensable for normal hemostasis. flTF is overexpressed in many forms of cancer and contributes to malignant growth; however, targeting flTF is risky due to the possibility of bleeding complications. We previously described that asTF is highly expressed in pancreatic ductal adenocarcinoma (PDAC) which account for ~75% of pancreatic cancer cases. Through non-canonical interactions with β1 integrins, asTF promotes activation of Akt and p42/44 MAPK, thus potentiating cancer cell migration and proliferation. We recently developed asTF-specific monoclonal antibody that inhibited growth and spread of PDAC in vivo in an orthotopic setting (Unruh et al, Oncotarget, in press). At this time it is not known whether, aside from PDAC, asTF also contributes to pathobiology of other pancreatic tumors such as pancreatic neuroendocrine tumors (pNET). Although pNETs comprise a minority subset of pancreatic cancer cases, pNET incidence is rapidly increasing and new therapies are thus needed for patients who ultimately progress on second line therapies which include mTOR inhibitors. Objective: To evaluate i) expression of asTF in pNET, and ii) potential utility of targeting intracellular pathways engaged by asTF in pNET. Methods: A tissue microarray containing 6 human pNET specimens was immunostained for asTF. Two human pNET cell lines, QGP1 and BON (a kind gift of Dr. Courtney Townsend, UTMB), were evaluated for TF isoform expression and their responsiveness to mTOR inhibition using the dual PI3K/mTOR ATP-site competitive inhibitor BEZ235. Results: All 6 pNET tissue specimens stained positive for asTF: 4 samples were highly positive and the remaining 2 moderately positive; normal pancreatic tissue specimens in the microarray exhibited minimal staining for asTF. qRT-PCR and western blotting analyses revealed that BON and QGP1 express flTF as well as asTF mRNA and protein, respectively. Compared to QGP1 cells, BON cells expressed ~3 fold more asTF and ~10 fold more flTF. Western blotting revealed that, compared to QGP1 cells, BON cells express significantly higher levels of β1 integrin. Treatment of QGP1 and BON cells with BEZ235 (400nM, 48 hours) markedly suppressed phosphorylation of mTOR targets including Akt, S6K1, 4E-BP1, and ULK1. Conclusion: To our knowledge, this is the first study to report that flTF and asTF are expressed in pNET tumor tissue and cultured cells. In pNET cell lines QGP1 and BON, BEZ235 effectively reduced phosphorylation of Akt, a downstream target of the pathways activated by asTF:β1 interactions. Further studies are warranted to evaluate asTF as a potential target in pNET, specifically strategies aimed at concurrent inhibition of asTF:β1 interactions and Akt/S6K1 activity. Citation Format: Clayton S. Lewis, Hala Elnakat Thomas, Fred V. Lucas, Vladimir Y. Bogdanov.{Authors}. Expression of alternatively spliced tissue factor in pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A39.

Combination effects of sorafenib with PI3K inhibitors under hypoxia in colorectal cancer.

AimThis study reports the influence of hypoxia on response of colorectal cancer cells to anticancer effects of sorafenib in combination with PI3K inhibitors GDC-0941 and BEZ-235.Materials and methodsAll hypoxic exposures were carried out at 1% O2/5% CO2. Antiproliferation activity was evaluated by 48 hours propidium iodide and 14 days clonogenic assay. Protein levels were evaluated by fluorescence ELISA. Metabolites lactate and glucose were evaluated biochemically.ResultsIn the 48-hour proliferation assay, sorafenib acted synergistically with GDC-0941 but not with BEZ-235. In long-term colony-forming assays, both GDC-0941 and BEZ-235 were shown to potentiate the antiproliferative activity of sorafenib. At the molecular level, the synergism is mediated through inhibition of pAKT, pS6, p4EBP1, pERK, cyclin D1, and Bcl-2. No change in hypoxia-inducible factor-1α (HIF-1α) levels was observed in cells treated with the combination of compounds under hypoxia. A significant reduction in glucose uptake and lactate release was observed in cells treated with the combination of compounds under normoxia and hypoxia.ConclusionCombinations of sorafenib with PI3K inhibitors BEZ-235 and GDC-0941 are efficacious under hypoxia. Thus, these anticancer combinations have a potential to overcome the hypoxia-mediated resistance mechanisms to antiproliferative agents in cancer therapy.

Functional exploration of colorectal cancer genomes using Drosophila.

The multigenic nature of human tumours presents a fundamental challenge for cancer drug discovery. Here we use Drosophila to generate 32 multigenic models of colon cancer using patient data from The Cancer Genome Atlas. These models recapitulate key features of human cancer, often as emergent properties of multigenic combinations. Multigenic models such as ras p53 pten apc exhibit emergent resistance to a panel of cancer-relevant drugs. Exploring one drug in detail, we identify a mechanism of resistance for the PI3K pathway inhibitor BEZ235. We use this data to identify a combinatorial therapy that circumvents this resistance through a two-step process of emergent pathway dependence and sensitivity we term ‘induced dependence'. This approach is effective in cultured human tumour cells, xenografts and mouse models of colorectal cancer. These data demonstrate how multigenic animal models that reference cancer genomes can provide an effective approach for developing novel targeted therapies.

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC50 on ATP concentration that allows prediction of the IC50 at different ATP concentrations in enzyme and cellular assays. Comparison of drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in a systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

Targeting PI3K/mTOR signaling exerts potent antitumor activity in pheochromocytoma in vivo

Pheochromocytomas (PCCs) are mostly benign tumors, amenable to complete surgical resection. However, 10-17% of cases can become malignant, and once metastasized, there is no curative treatment for this disease. Given the need to identify the effective therapeutic approaches for PCC, we evaluated the antitumor potential of the dual-PI3K/mTOR inhibitor BEZ235 against these tumors. We employed an in vivo model of endogenous PCCs (MENX mutant rats), which closely recapitulate the human tumors. Mutant rats with PCCs were treated with 2 doses of BEZ235 (20 and 30 mg/kg), or with placebo, for 2 weeks. Treatment with BEZ235 induced cytostatic and cytotoxic effects on rat PCCs, which could be appreciated by both staining the tumors ex vivo with appropriate markers and non-invasively by functional imaging (diffusion-weighted magnetic resonance imaging) in vivo Transcriptomic analyses of tumors from rats treated with BEZ235 or placebo-identified potential mediators of therapy response were performed. Slc6a2, encoding the norepinephrine transporter (NET), was downregulated in a dose-dependent manner by BEZ235 in rat PCCs. Moreover, BEZ235 reduced Slc6a2/NET expression in PCC cell lines (MPC) also. Studies of a BEZ235-resistant derivative of the MPC cell line confirmed that the reduction of NET expression associates with the response to the drug. Reduction of NET expression after BEZ235 treatment in vivo could be monitored by positron emission tomography (PET) using a tracer targeting NET. Altogether, here we demonstrate the efficacy of BEZ235 against PCC in vivo, and show that functional imaging can be employed to monitor the response of PCC to PI3K/mTOR inhibition therapy.

Insulin elevates leptin secretion and mRNA levels via cyclic AMP in 3T3-L1 adipocytes deprived of glucose.

AimsLeptin plays an important role in the pathogenesis of obesity and diabetes, yet the regulatory mechanisms of this hormone have not been fully elucidated. In this study, we aimed to clarify the roles of insulin and glucose in leptin secretion and mRNA production using inhibitors of insulin signal transduction in adipocytes cultured under glucose-free or normal conditions.MethodsDifferentiated 3T3-L1 adipocytes were stimulated with insulin in combination with inhibitors for phosphoinositide 3-kinase (PI3K), Akt, and phosphodiesterase 3B (PDE3B), as well as epinephrine and a cyclic AMP (cAMP) analog under glucose-free or normal conditions. After 8 h of stimulation, leptin protein levels in the media and leptin mRNA expression levels in the adipocytes were measured.ResultsInsulin significantly increased the secretion and mRNA levels of leptin under the depletion of glucose. Glucose augmented basal leptin secretion without insulin, while glucose nullified insulin-induced leptin mRNA upregulation. The PI3K inhibitor BEZ-235, the Akt inhibitor MK-2206, and the PDE3B inhibitor cilostazol attenuated the insulin stimulation of leptin secretion, but did not suppress the insulin-induced leptin mRNA upregulation with glucose depletion. In contrast to the glucose-free condition, insulin failed to upregulate leptin mRNA in the presence of glucose. The cAMP analog dibutyryl cAMP and epinephrine decreased both leptin secretion and mRNA regardless of glucose supplementation.ConclusionInsulin alone stimulates leptin secretion and elevates leptin mRNA levels via cAMP under the lack of glucose metabolism, while glucose is a significant and ambivalent effector on the insulin effects of leptin.

Targeting of PI3K/AKT/mTOR pathway to inhibit T cell activation and prevent graft-versus-host disease development.

BackgroundGraft-versus-host disease (GvHD) remains the major obstacle to successful allogeneic hematopoietic stem cell transplantation, despite of the immunosuppressive regimens administered to control T cell alloreactivity. PI3K/AKT/mTOR pathway is crucial in T cell activation and function and, therefore, represents an attractive therapeutic target to prevent GvHD development. Recently, numerous PI3K inhibitors have been developed for cancer therapy. However, few studies have explored their immunosuppressive effect.MethodsThe effects of a selective PI3K inhibitor (BKM120) and a dual PI3K/mTOR inhibitor (BEZ235) on human T cell proliferation, expression of activation-related molecules, and phosphorylation of PI3K/AKT/mTOR pathway proteins were analyzed. Besides, the ability of BEZ235 to prevent GvHD development in mice was evaluated.ResultsSimultaneous inhibition of PI3K and mTOR was efficient at lower concentrations than PI3K specific targeting. Importantly, BEZ235 prevented naïve T cell activation and induced tolerance of alloreactive T cells, while maintaining an adequate response against cytomegalovirus, more efficiently than BKM120. Finally, BEZ235 treatment significantly improved the survival and decreased the GvHD development in mice.ConclusionsThese results support the use of PI3K inhibitors to control T cell responses and show the potential utility of the dual PI3K/mTOR inhibitor BEZ235 in GvHD prophylaxis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0343-5) contains supplementary material, which is available to authorized users.

Superior efficacy of co-treatment with the dual PI3K/mTOR inhibitor BEZ235 and histone deacetylase inhibitor Trichostatin A against NSCLC.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. NSCLC development and progression have recently been correlated with the heightened activation of histone deacetylases (HDACs) and PI3K/Akt signaling pathways. Targeted inhibition of these proteins is promising approach for the development of novel therapeutic strategies to treat patients with advanced NSCLC. For this reason, we combined a dual PI3K and mTOR inhibitor, BEZ235 with the HDAC inhibitor Trichostatin A (TSA), to determine their combined effects on human NSCLC. In this study, we initially discovered that co-treatment with BEZ235 and TSA showed a synergistic effect on inhibition of NSCLC cell proliferation and induction of apoptosis. The combination treatment also synergistically suppressed NSCLC migration, invasion and the NSCLC epithelial-mesenchymal transition (EMT) in vitro. The synergistic effect was also evidenced by declines in xenograft growth and metastasis rates and in ki-67 protein expression in vivo. Together, these results indicated that BEZ235 and TSA combination treatment significantly increased anti-tumor activities compared with BEZ235 and TSA alone, supporting a further evaluation of combination treatment for NSCLC.

Abstract 4391: Overcoming primary ibrutinib resistance in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets. Bruton's tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice. Citation Format: Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. Overcoming primary ibrutinib resistance in mantle cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4391.

Abstract 4666: Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts

Abstract Background: There is an urgent need for improved therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. The Phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in HNSCC, particularly in human papillomavirus (HPV) positive disease. PI3K pathway inhibitors are under active investigation in preclinical models and clinical trials, however it is not clear which patient population will benefit most from these agents. Methods: Thirty unique HNSCC cell lines including five HPV positive lines were characterized with whole exome sequencing and copy number arrays. All lines were treated over a 10-point dose range with a PIK3CA specific inhibitor (BYL719), a pan PI3K inhibitor (GDC-0941), a combined pan-PI3k and mTOR inhibitor (BEZ-235), mTOR inhibitor (everolimus) and a combination of BYL719 and everolimus. Five unique patient derived xenografts (PDX) were treated with BYL719 and everolimus alone and in combination. Results: The selective PIK3CA inhibitor BYL719 was preferentially active in cell lines with PIK3CA activating mutations and amplifications (&amp;gt;100% relative PIK3CA amplification), however HRAS mutant lines were resistant. These genomic markers of sensitivity and resistance did not apply to the pan-PI3K inhibitor GDC-0941 or combined PI3K-mTOR inhibitor BEZ-235, however these agents were more active in HPV-positive cell lines (P&amp;lt;0.05). BYL719 and everolimus were individually effective at decreasing tumor growth in four PDX models including a model with an activating PIK3CA mutation, two PIK3CA amplified models and a PIK3CA wild-type model. Combined PIK3CA and mTOR inhibition appeared to be synergistic and evolved resistance never developed to this treatment, however the combination caused significant weight loss in the PDX models. Interpretation: PI3K inhibitors were broadly active in HNSCC cell lines and PDXs and their application should not be solely restricted to tumors with activating PIK3CA mutations or amplifications. Agents with broader isoform activity and combined PI3K and mTOR treatment were more potent, however combined inhibitor treatment appeared toxic in the PDX models. This trade off of toxicity and efficacy needs to balanced in the clinical setting. Citation Format: Anthony C. Nichols, Laurie Ailkes, Ren Sun, Morgan Black, Alessandro Datti, Frederick Vizeacoumar, Nicole Pinto, Kara Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, Joe Mymryk, Paul C. Boutros, John W. Barrett. Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4666.

Abstract 2468: Combined targeting of the EWS/ETS transcriptional program by BET bromodomain and PI3K pathway inhibition blocks tumorigenicity and increases apoptosis in Ewing sarcoma

Abstract Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations that give rise to chimeric proteins (EWS-ETS), which generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor (BEZ235) of the PI3K pathway. Microarray analysis revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference of BRD3 or BRD4 expression but not by BRD2 blockade, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. JQ1 treatment not only suppressed an ES specific expression profile but also blocked contact dependent and independent proliferation of different ES lines. But subsequent analysis of proliferation after transient knockdown of either BRD3 or BRD4 did not recapitulate inhibition of proliferation as observed after JQ1 treatment, indicating the necessary simultaneous blockade of both proteins. However, inhibition of proliferation after JQ1 treatment was due to a partial G1 arrest and S phase elongation of the cell cycle. In addition, induction of apoptosis as demonstrated by PARP1-, CASP7-cleavage and increased CASP3 activity significantly contributed to the reduction of the proliferative ability of ES lines. Single or combination treatment with the PI3K/mTOR inhibitor BEZ235 increased apoptosis of ES cell lines although single treatment with BEZ235 was less effective than JQ1 application. Consequently, tumor development was dose dependently suppressed with increased formation of apoptotic bodies in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program. Citation Format: Tim Hensel, Chiara Giorgi, Fiona Becker-Dettling, Julia Calzada-Wack, Frauke Neff, Thorsten Buch, Oxana Schmidt, Beat W. Schäfer, Stefan Burdach, Günther HS Richter. Combined targeting of the EWS/ETS transcriptional program by BET bromodomain and PI3K pathway inhibition blocks tumorigenicity and increases apoptosis in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2468.