Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

Malignant pleural mesothelioma (MPM) originates in most of the cases from chronic inflammation of the mesothelium due to exposure to asbestos fibers. Given the limited effect of chemotherapy, a big effort is being made to find new treatment options. The PI3K/mTOR pathway was reported to be upregulated in MPM. We tested the cell growth inhibition properties of two dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 on 19 MPM cell lines. We could identify resistant and sensitive lines; however, there was no correlation to the downregulation of PI3K/mTOR activity markers. As a result of mTOR inhibition, both drugs efficiently induced long-term autophagy but not cell death. Autophagy blockade by chloroquine in combination with the dual PI3K/mTOR inhibitors significantly induced caspase-independent cell death involving RIP1 in the sensitive cell line SPC212. Cell death in the resistant cell line Mero-82 was less pronounced, and it was not induced via RIP1-dependent mechanism, suggesting the involvement of RIP1 downstream effectors. Cell death induction was confirmed in 3D systems. Based on these results, we identify autophagy as one of the main mechanisms of cell death resistance against dual PI3K/mTOR inhibitors in MPM. As PI3K/mTOR inhibitors are under investigation in clinical trials, these results may help interpreting their outcome and suggest ways for intervention.Malignant pleural mesothelioma (MPM) is sensitive to phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling inhibitors due to the activation of PI3K/mTOR signaling.^{1, 2} The activation may result from inactivation of INP4A phosphatase, which is downregulated in 44% of MPM (presented at IMIG2014), or alterations in PI3K signaling components, which are mutated in 9% of MPM,³ while receptor tyrosine kinase mutations/amplifications have not been identified in two recent high-throughput studies.^{4, 5}One of the tumor-suppressor genes frequently mutated in MPM is NF2 and NF2-null cells were shown to be sensitive to growth-inhibitory effects of rapamycin⁶ via mechanisms involving PI3K signaling-independent mTORC1 activation. However, the mTOR inhibitor, everolimus, showed no therapeutic benefit in unselected MPM patients.⁷ As mTORC1 inhibitors often lead to a feedback activation of PI3K activation in cancers,^{8, 9} we postulated that dual PI3K–mTOR inhibitors may yield greater therapeutic benefit. Furthermore, NF2 was also shown to inhibit PI3K activity by binding to PI3K enhancer-L (PIKE-L), which disrupts binding of PIKE-L to PI3K¹⁰ and loss of NF2 in schwannoma was shown to sensitize to PI3K inhibitors.¹¹In a screen on the dual PI3K/mTOR inhibitor NVP-BEZ235, within the Sanger Institute/MGH''s ‘Genomics of Drug Sensitivity'' screening panel,¹² CDKN2A deletion was shown to be associated with increased sensitivity. Because NF2 and CDKN2A are indeed the genes most frequently mutated in MPM, blocking PI3K/mTOR signaling might be a valid approach to circumvent the difficulty of applying targeted therapy in the absence of an identified oncogene. The rationale for targeting the PI3K/mTOR pathway is also supported by the association of increased activity with a worse clinical outcome.^{13, 14}NVP-BEZ235^(ref15) and GDC-0980^(ref16) are small-molecule inhibitors of class I PI3K and mTOR (mTORC1 and mTORC2). GDC-0980 has been tested in phase I studies where the phase I extension cohort showed two objective responses among 26 patients with mesothelioma.¹⁷ Despite these encouraging results, this drug will not be explored further because of side effects observed in another clinical trial.¹⁸ This, however, should not deter us for trying to find means to improve the antitumor effect of this class of agents. We have previously shown that PI3K/mTOR signaling inhibition sensitizes mesothelioma cells to drugs that are effluxed via ABCG2 transporter by inhibiting the function of ABCG2.¹⁹ In this study, we aimed at identifying the underlying mechanisms responsible for sensitivity versus resistance towards PI3K/mTOR inhibition in a large panel of mesothelioma cell lines. We observed that PI3K/mTOR inhibition increases autophagic rate, which constitutes an efficient mechanism of resistance by inducing growth arrest and survival. However, blocking autophagy, which per se affects cell growth, is synthetically lethal when combined with PI3K/mTOR inhibitors by a mechanism involving receptor-interacting protein kinase 1 (RIP1)-dependent cell death.     

Autophagy Correlates with the Therapeutic Responsiveness of Malignant Pleural Mesothelioma in 3D Models

Malignant pleural mesothelioma is a highly chemoresistant solid tumor. We have studied this apoptotic resistance using in vitro and ex vivo three-dimensional models, which acquire a high level of chemoresistance that can be reduced by PI3K/mTOR inhibitors. Here, we investigate the activity of GDC-0980, a novel dual PI3K/mTOR inhibitor, which has been proposed to be effective in mesothelioma. In this work, we aimed to identify mechanisms and markers of efficacy for GDC-0980 by utilizing 3D models of mesothelioma, both in vitro multicellular spheroids and ex vivo tumor fragment spheroids grown from patient tumor samples. We found that a subset of mesothelioma spheroids is sensitive to GDC-0980 alone and to its combination with chemotherapy. Unexpectedly, this sensitivity did not correlate with the activation of the Akt/mTOR pathway. Instead, sensitivity to GDC-0980 correlated with the presence of constitutive ATG13 puncta, a feature of autophagy, a cellular program that supports cells under stress. In tumor fragment spheroids grown from 21 tumors, we also found a subset (n = 11) that was sensitive to GDC-0980, a sensitivity that also correlated with the presence of ATG13 puncta. Interference with autophagy by siRNA of ATG7, an essential autophagic protein, increased the response to chemotherapy, but only in the sensitive multicellular spheroids. In the spheroids resistant to GDC-0980, autophagy appeared to play no role. In summary, we show that GDC-0980 is effective in mesothelioma 3D models that display ATG13 puncta, and that blockade of autophagy increases their response to chemotherapy. For the first time, we show a role for autophagy in the response to chemotherapy of 3D models of mesothelioma and propose ATG13 as a potential biomarker of the therapeutic responsiveness of mesothelioma.     

Genotype Directed Therapy in Murine Mismatch Repair Deficient Tumors

The PI3K/AKT/mTOR pathway has frequently been found activated in human tumors. We show that in addition to Wnt signaling dysfunction, the PI3K/AKT/mTOR pathway is often upregulated in mouse Msh2^−/− initiated intestinal tumors. NVP-BEZ235 is a dual PI3K/mTOR inhibitor toxic to many cancer cell lines and currently involved in clinical trials. We have treated two mouse models involving Msh2 that develop small intestinal and/or colonic tumors with NVP-BEZ235, and a subset of animals with NVP-BEZ235 and MEK inhibitor ADZ4266. The disease phenotype has been followed with pathology, ¹⁸F FDG PET imaging, and endoscopy. Intestinal adenocarcinomas are significantly decreased in multiplicity by both drug regimens. The majority of tumors treated with combined therapy regress significantly, while a small number of highly progressed tumors persist. We have examined PTEN, AKT, MEK 1&2, MAPK, S6K, mTOR, PDPK1, and Cyclin D1 and find variable alterations that include downregulation of PTEN, upregulation of AKT and changes in its phosphorylated forms, upregulation of pMEK 1&2, p42p44MAPK, pS6K, and Cyclin D1. Apoptosis has been found intact in some tumors and not in others. Our data indicate that NVP-BEZ235 alone and in combination with ADZ4266 are effective in treating a proportion of colorectal cancers, but that highly progressed resistant tumors grow in the presence of the drugs. Pathways upregulated in some resistant tumors also include PDPK1, suggesting that metabolic inhibitors may also be useful in treating these tumors.     

The synergistic antitumor effect of IL-6 neutralization with NVP-BEZ235 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) still ranks among the top cancers worldwide with high incidence and mortality. Due to abnormal activation of the PI3K/AKT/mTOR signalling pathway in HCC, targeting this pathway represents a potential therapeutic strategy. NVP-BEZ235 is a novel dual-targeted ATP-competitive PI3K/mTOR inhibitor that has shown effective antitumor effects. In this study, we found that interleukin-6 (IL-6) was significantly increased after exposure to NVP-BEZ235, and we proposed a treatment in which an anti-IL-6 antibody was combined with NVP-BEZ235 for HCC. In vitro results revealed that targeted inhibition of IL-6 potentiated the antitumor effects of NVP-BEZ235 in HCC cells. The mechanism might be attributed to their synergistic inhibitory activity on the PI3K/AKT/mTOR signalling pathway. Furthermore, an in vivo study demonstrated that combined administration of NVP-BEZ235 and anti-IL-6 Ab reduced HCC tumour load more effectively than either NVP-BEZ235 or anti-IL-6 Ab treatment alone. These findings add guidance value to the analysis of HCC and provide a reference for clinical treatment.Subject terms: Targeted therapies, Liver cancer     

Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 Has a Therapeutic Potential and Sensitizes Cisplatin in Nasopharyngeal Carcinoma

Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells and thus has been considered as a promising drug target. To ascertain a therapeutical approach of nasopharyngeal carcinoma (NPC), we hypothesized NVP-BEZ235, a novel and potent imidazo[4,5-c] quinolone derivative, that dually inhibits both PI3K and mTOR kinases activities, had antitumor activity in NPC. Expectedly, we found that NVP-BEZ235 selectively inhibited proliferation of NPC cells rather than normal nasopharyngeal cells using MTT assay. In NPC cell lines, with the extended exposure, NVP-BEZ235 selectively inhibited proliferation of NPC cells harboring PIK3CA mutation, compared to cells with wild-type PIK3CA. Furthermore, exposure of NPC cells to NVP-BEZ235 resulted in G1 growth arrest by Propidium iodide uptake assay, reduction of cyclin D1and CDK4, and increased levels of P27 and P21 by Western blotting, but negligible apoptosis. Moreover, we found that cisplatin (CDDP) activated PI3K/AKT and mTORC1 pathways and NVP-BEZ235 alleviated the activation by CDDP through dually targeting PI3K and mTOR kinases. Also, NVP-BEZ235 combining with CDDP synergistically inhibited proliferation and induced apoptosis in NPC cells. In CNE2 and HONE1 nude mice xenograft models, orally NVP-BEZ235 efficiently attenuated tumor growth with no obvious toxicity. In combination with NVP-BEZ235 and CDDP, there was dramatic synergy in shrinking tumor volumes and inducing apoptosis through increasing Noxa, Bax and decreasing Mcl-1, Bcl-2. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential as a monotherapy or in combination with CDDP for NPC treatment.     

Deciphering Combinations of PI3K/AKT/mTOR Pathway Drugs Augmenting Anti-Angiogenic Efficacy In Vivo

Ocular neovascularization is a common pathology associated with human eye diseases e.g. age-related macular degeneration and proliferative diabetic retinopathy. Blindness represents one of the most feared disabilities and remains a major burden to health-care systems. Current approaches to treat ocular neovascularisation include laser photocoagulation, photodynamic therapy and anti-VEGF therapies: Ranibizumab (Lucentis) and Aflibercept (Eylea). However, high clinical costs, frequent intraocular injections, and increased risk of infections are challenges related with these standards of care. Thus, there is a clinical need to develop more effective drugs that overcome these challenges. Here, we focus on an alternative approach by quantifying the in vivo anti-angiogenic efficacy of combinations of phosphatidylinositol-3-kinase (PI3K) pathway inhibitors. The PI3K/AKT/mTOR pathway is a complex signalling pathway involved in crucial cellular functions such as cell proliferation, migration and angiogenesis. RT-PCR confirms the expression of PI3K target genes (pik3ca, pik3r1, mtor and akt1) in zebrafish trunks from 6 hours post fertilisation (hpf) and in eyes from 2 days post fertilisation (dpf). Using both the zebrafish intersegmental vessel and hyaloid vessel assays to measure the in vivo anti-angiogenic efficacy of PI3K/Akt/mTOR pathway inhibitors, we identified 5 µM combinations of i) NVP-BEZ235 (dual PI3K-mTOR inhibitor) + PI-103 (dual PI3K-mTOR inhibitor); or ii) LY-294002 (pan-PI3K inhibitor) + NVP-BEZ235; or iii) NVP-BEZ235 + rapamycin (mTOR inhibitor); or iv) LY-294002 + rapamycin as the most anti-angiogenic. Treatment of developing larvae from 2–5 dpf with 5 µM NVP-BEZ235 plus PI-103 resulted in an essentially intact ocular morphology and visual behaviour, whereas other combinations severely disrupted the developing retinal morphology and visual function. In human ARPE19 retinal pigment epithelium cells, however, no significant difference in cell number was observed following treatment with the inhibitor combinations. Collectively, these results highlight the potential of combinations of PI3K/AKT/mTOR pathway inhibitors to safely and effectively treat ocular neovascularization.     

Multi-level targeting of the phosphatidylinositol-3-kinase pathway in non-small cell lung cancer cells

Introduction

We assessed expression of p85 and p110α PI3K subunits in non-small cell lung cancer (NSCLC) specimens and the association with mTOR expression, and studied effects of targeting the PI3K/AKT/mTOR pathway in NSCLC cell lines.

Methods

Using Automated Quantitative Analysis we quantified expression of PI3K subunits in two cohorts of 190 and 168 NSCLC specimens and correlated it with mTOR expression. We studied effects of two PI3K inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and NVP-BKM120, alone and in combination with rapamycin in 6 NSCLC cell lines. We assessed activity of a dual PI3K/mTOR inhibitor, NVP-BEZ235 alone and with an EGFR inhibitor.

Results

p85 and p110α tend to be co-expressed (p<0.001); p85 expression was higher in adenocarcinomas than squamous cell carcinomas. High p85 expression was associated with advanced stage and poor survival. p110α expression correlated with mTOR (ρ = 0.276). In six NSCLC cell lines, addition of rapamycin to {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or NVP-BKM120 was synergistic. Even very low rapamycin concentrations (1 nM) resulted in sensitization to PI3K inhibitors. NVP-BEZ235 was highly active in NSCLC cell lines with IC₅₀s in the nanomolar range and resultant down-regulation of pAKT and pP70S6K. Adding Erlotinib to NVP-BEZ235 resulted in synergistic growth inhibition.

Conclusions

The association between PI3K expression, advanced stage and survival in NSCLC suggests that it might be a valuable drug target. Concurrent inhibition of PI3K and mTOR is synergistic in vitro, and a dual PI3K/mTOR inhibitor was highly active. Adding EGFR inhibition resulted in further growth inhibition. Targeting the PI3K/AKT/mTOR pathway at multiple levels should be tested in clinical trials for NSCLC.     

Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule

Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G₁ phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G₂/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.     

Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis

Background

Aberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear.

Methods

PubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model.

Results

Analysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13).

Conclusion

Activation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway.     

Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n?=?9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n?=?4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.     

Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma

Breaking resistance to chemotherapy is a major goal of combination therapy in many tumors, including advanced neuroblastoma. We recently demonstrated that increased activity of the PI3K/Akt network is associated with poor prognosis, thus providing an ideal target for chemosensitization. Here we show that targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells. Importantly, this increase in apoptosis was dependent on scheduling: while pretreatment with the inhibitor reduced doxorubicin-induced apoptosis, the sensitizing effect in co-treatment could further be increased by delayed addition of the inhibitor post chemotherapy. Desensitization for doxorubicin-induced apoptosis seemed to be mediated by a combination of cell cycle-arrest and autophagy induction, whereas sensitization was found to occur at the level of mitochondria within one hour of NVP-BEZ235 posttreatment, leading to a rapid loss of mitochondrial membrane potential with subsequent cytochrome c release and caspase-3 activation. Within the relevant time span we observed marked alterations in a ∼30 kDa protein associated with mitochondrial proteins and identified it as VDAC1/Porin protein, an integral part of the mitochondrial permeability transition pore complex. VDAC1 is negatively regulated by the PI3K/Akt pathway via GSK3β and inhibition of GSK3β, which is activated when Akt is blocked, ablated the sensitizing effect of NVP-BEZ235 posttreatment. Our findings show that cancer cells can be sensitized for chemotherapy induced cell death – at least in part – by NVP-BEZ235-mediated modulation of VDAC1. More generally, we show data that suggest that sequential dosing, in particular when multiple inhibitors of a single pathway are used in the optimal sequence, has important implications for the general design of combination therapies involving molecular targeted approaches towards the PI3K/Akt/mTOR signaling network.     

Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.     

Relationships between Signaling Pathway Usage and Sensitivity to a Pathway Inhibitor: Examination of Trametinib Responses in Cultured Breast Cancer Lines

Cellular signaling pathways involving mTOR, PI3K and ERK have dominated recent studies of breast cancer biology, and inhibitors of these pathways have formed a focus of numerous clinical trials. We have chosen trametinib, a drug targeting MEK in the ERK pathway, to address two questions. Firstly, does inhibition of a signaling pathway, as measured by protein phosphorylation, predict the antiproliferative activity of trametinib? Secondly, do inhibitors of the mTOR and PI3K pathways synergize with trametinib in their effects on cell proliferation? A panel of 30 human breast cancer cell lines was chosen to include lines that could be classified according to whether they were ER and PR positive, HER2 over-expressing, and “triple negative”. Everolimus (targeting mTOR), NVP-BEZ235 and GSK2126458 (both targeting PI3K/mTOR) were chosen for combination experiments. Inhibition of cell proliferation was measured by IC₅₀ values and pathway utilization was measured by phosphorylation of signaling kinases. Overall, no correlation was found between trametinib IC₅₀ values and inhibition of ERK signaling. Inhibition of ERK phosphorylation was observed at trametinib concentrations not affecting proliferation, and sensitivity of cell proliferation to trametinib was found in cell lines with low ERK phosphorylation. Evidence was found for synergy between trametinib and either everolimus, NVP-BEZ235 or GSK2126458, but this was cell line specific. The results have implications for the clinical application of PI3K/mTOR and MEK inhibitors.     

Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (³¹P)- and proton (¹H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.     

NVP-BEZ235, a dual PI3K/mTOR inhibitor,induces cell death through alternate routes in prostate cancer cells depending on the PTEN genotype

Deregulation of the PI3K-AKT/mTOR pathway due to mutation of the tumor suppressor gene PTEN frequently occurs in human prostate cancer and is therefore considered to be an attractive therapeutic target. Here, we investigated how the PTEN genotype affected the antitumor effect of NVP-BEZ235 in human prostate cancer cells. In this setting, NVP-BEZ235 induced cell death in a PTEN-independent manner. NVP-BEZ235 selectively induced apoptotic cell death in the prostate cancer cell line DU145, which harbors wild-type PTEN; however, in the PC3 cell line, which is PTEN-null, treatment with NVP-BEZ235 resulted in autophagic cell death. Consistently, NVP-BEZ235 treatment did not result in the cleavage of caspase-3; instead, it resulted in the conversion of LC3-I to LC3-II, indicating autophagic cell death; these results suggest that an alternate mechanism of cell death is induced by NVP-BEZ235 in PTEN-null prostate cancer cells. Based on our findings, we conclude that the PTEN/PI3K/Akt pathway is critical for prostate cancer survival, and targeting PI3K signaling by NVP-BEZ235 may be beneficial in the treatment of prostate cancer, independent of the PTEN genotype.     

The PI3-kinase/mTOR-targeting drug NVP-BEZ235 inhibits growth and IgE-dependent activation of human mast cells and basophils

The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring (3)H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC(50) values in the HMC-1.1 subclone lacking KIT D816V (0.025 μM) and the HMC-1.2 subclone expressing KIT D816V (0.005 μM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1(nu) mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC(50) 0.5-1 μM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.     

Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor,ARQ 092

As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies.     

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer

Purpose

To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).

Experimental Design

PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.

Results

In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC₅₀ = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).

Conclusions

These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.     

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.