pi103 and Glioblastoma

Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells.. Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by γH2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations.. We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (~ 80% vs. ~ 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells.. The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism.

Topics: Brain Neoplasms; Cell Line, Tumor; Chemoradiotherapy; DNA-Activated Protein Kinase; Furans; Glioblastoma; Humans; Phosphatidylinositol 3-Kinases; Pyridines; Pyrimidines; Radiation Tolerance; TOR Serine-Threonine Kinases

Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.. Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition.. We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy.. Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered.

Topics: Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; DNA Damage; Furans; Gene Expression Regulation, Neoplastic; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyridines; Pyrimidines; Radiation Tolerance; Radiation-Sensitizing Agents; Single-Cell Analysis; TOR Serine-Threonine Kinases

High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

Topics: Actin Cytoskeleton; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cytoskeleton; Furans; Glioblastoma; HSP90 Heat-Shock Proteins; Humans; Isoxazoles; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Pyrimidines; Resorcinols; TOR Serine-Threonine Kinases

Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Dacarbazine; Furans; Glioblastoma; Humans; Lactic Acid; Phosphorylcholine; Proton Magnetic Resonance Spectroscopy; Pyridines; Pyrimidines; Temozolomide; Treatment Outcome

Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Colonic Neoplasms; DNA Damage; Drug Administration Schedule; Drug Synergism; Furans; Glioblastoma; HSP90 Heat-Shock Proteins; Humans; Isoxazoles; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Pyrimidines; Radiation Tolerance; Radiation-Sensitizing Agents; Resorcinols; TOR Serine-Threonine Kinases; Up-Regulation

Glioblastoma multiforme (GBM) is the most common primary brain tumor and among the most difficult to treat malignancies per se. In almost 90% of all GBM alterations in the PI3K/Akt/mTOR have been found, making this survival cascade a promising therapeutic target, particular for combination therapy that combines an apoptosis sensitizer, such as a pharmacological inhibitor of PI3K, with an apoptosis inducer, such as radio- or chemotherapy. However, while in vitro data focusing mainly on established cell lines has appeared rather promising, this has not translated well to a clinical setting. In this study, we analyze the effects of the dual kinase inhibitor PI-103, which blocks PI3K and mTOR activity, on three matched pairs of GBM stem cells/differentiated cells. While blocking PI3K-mediated signaling has a profound effect on cellular proliferation, in contrast to data presented on two GBM cell lines (A172 and U87) PI-103 actually counteracts the effect of chemotherapy. While we found no indications for a potential role of the PI3K signaling cascade in differentiation, we saw a clear and strong contribution to cellular motility and, by extension, invasion. While blocking PI3K-mediated signaling concurrently with application of chemotherapy does not appear to be a valid treatment option, pharmacological inhibitors, such as PI-103, nevertheless have an important place in future therapeutic approaches.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dacarbazine; Drug Synergism; Furans; Glioblastoma; Humans; Neoplasm Staging; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Pyridines; Pyrimidines; Signal Transduction; Temozolomide; TOR Serine-Threonine Kinases

The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild-type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3-kinase (PI3-K)/AKT pathway-focused gene profiles were examined by real-time polymerase chain reaction-based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen-activated protein kinase kinase inhibitor U0126. The dual PI3-K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal-regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.

Topics: Butadienes; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Furans; Gene Expression; Glioblastoma; Humans; Nitriles; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyridines; Pyrimidines; Quinazolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Amplification of the gene encoding the epidermal growth factor receptor (EGFR) occurs commonly in glioblastoma (GBM), leading to activation of downstream kinases, including phosphatidylinositol 3'-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). A serine-threonine kinase, mTOR controls cell growth by regulating mRNA translation, metabolism, and autophagy; acting as both a downstream effector and upstream regulator of PI3K. These signaling functions are distributed between at least two distinct complexes, mTORC1 and mTORC2 with respect to pathway specificity. We have investigated mTOR signaling in glioma cells with the allosteric mTORC1 inhibitor rapamycin, the mTORC1/2 inhibitor Ku-0063794, a dual PI3K/mTORC1/2 kinase inhibitor PI-103, and siRNA against raptor, rictor, or mTOR, and evaluated the value of mTOR inhibitors for the treatment of glioblastoma.

Topics: Adaptor Proteins, Signal Transducing; Carrier Proteins; Cell Line, Tumor; ErbB Receptors; Furans; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiprotein Complexes; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proteins; Pyridines; Pyrimidines; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.

Topics: Blotting, Western; Brain; Endothelial Cells; Flow Cytometry; Furans; Glioblastoma; Humans; Microscopy, Fluorescence; Pyridines; Pyrimidines; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases; Transfection

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110alpha with IC(50) < or = 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials.

Topics: Administration, Oral; Animals; Cell Proliferation; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Female; Furans; Glioblastoma; Humans; Indazoles; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Pyridines; Pyrimidines; Sulfonamides; Thiophenes; TOR Serine-Threonine Kinases; Umbilical Veins; Xenograft Model Antitumor Assays

The failure of conventional therapies in glioblastoma (GBM) is largely due to an aberrant activity of survival cascades, such as PI3 kinase (PI3K)/Akt-mediated signaling. This study is the first to show that the class I PI3K inhibitor, PI-103, enhances chemotherapy-induced cell death of GBM cells. Concurrent treatment with PI-103 and DNA-damaging drugs, in particular doxorubicin, significantly increases apoptosis and reduces colony formation compared with chemotherapy treatment alone. The underlying molecular mechanism for this chemosensitization was shown by two independent approaches, that is, pharmacological and genetic inhibition of PI3K, DNA-PK and mTOR, to involve inhibition of DNA-PK-mediated DNA repair. Accordingly, blockage of PI3K or DNA-PK, but not of mTOR, significantly delays the resolution of doxorubicin-induced DNA damage and concomitantly increases apoptosis. Importantly, not only are several GBM cell lines chemosensitized by PI-103 but also GBM stem cells. Clinical relevance was further confirmed by the use of primary cultured GBM cells, which also exhibit increased cell death and reduced colony formation on combined treatment with PI-103 and doxorubicin. By identifying class I PI3K inhibitors as powerful agents in enhancing the lethality of DNA-damaging drugs, to which GBMs are usually considered unresponsive, our findings have important implications for the design of rational combination regimens in overcoming the frequent chemoresistance of GBM.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Chromones; DNA Damage; DNA Repair; DNA-Activated Protein Kinase; Doxorubicin; Furans; Glioblastoma; Histones; Humans; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Pyrimidines; Signal Transduction

The phosphoinositide 3'-kinase (PI3K)-mediated signaling pathway plays a key role in fundamental cellular functions important in normal cellular homeostasis and malignant transformation. Deregulated signaling through this pathway contributes to development of gliomas and their resistance to radiation and chemotherapy. Targeting the PI3K signaling pathway has thus emerged as a promising approach to successful treatment of gliomas. We assessed the radiosensitizing potential of four small-molecule inhibitors that differ in their activities against specific isoforms of the PI3K 110-kDa catalytic subunit (p110). p110alpha inhibitors blocked phosphorylation of both protein kinase B/Akt and S6 in all cell lines examined, effectively decreased cellular proliferation, and produced additive cytotoxic effects in combination with radiation therapy. The p110beta inhibitor exhibited limited biochemical effects and failed to decrease cellular proliferation or viability as either a single agent or in combination with radiation or rapamycin. In vivo studies examining the effects of the p110alpha inhibitor in combination with radiation indicated a significant reduction in tumor growth rate induced by the combined treatment compared with each treatment modality alone. This translated into a trend toward prolonged time-to-failure for mice in the combination treatment group. In conclusion, PI3K inhibitors are promising agents in the treatment of glioblastomas, especially when used in combination with ionizing radiation.

Topics: Aniline Compounds; Animals; Apoptosis; Astrocytes; Blotting, Western; Brain Neoplasms; Cell Proliferation; Cells, Cultured; Chromones; Combined Modality Therapy; Furans; Glioblastoma; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Isoforms; PTEN Phosphohydrolase; Pyridines; Pyrimidines; Radiation, Ionizing; Xenograft Model Antitumor Assays