mk-2206 and Glioblastoma

PD-L1 up-regulation in cancer contributes to immune evasion by tumor cells. Here, we show that Wnt ligand and activated EGFR induce the binding of the β-catenin/TCF/LEF complex to the CD274 gene promoter region to induce PD-L1 expression, in which AKT activation plays an important role. β-Catenin depletion, AKT inhibition, or PTEN expression reduces PD-L1 expression in tumor cells, enhances activation and tumor infiltration of CD8+ T cells, and reduces tumor growth, accompanied by prolonged mouse survival. Combined treatment with a clinically available AKT inhibitor and an anti-PD-1 antibody overcomes tumor immune evasion and greatly inhibits tumor growth. In addition, AKT-mediated β-catenin S552 phosphorylation and nuclear β-catenin are positively correlated with PD-L1 expression and inversely correlated with the tumor infiltration of CD8+ T cells in human glioblastoma specimens, highlighting the clinical significance of β-catenin activation in tumor immune evasion.

Topics: Allografts; Animals; Antibodies; B7-H1 Antigen; beta Catenin; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Transcription, Genetic; Transfection; Tumor Burden; Tumor Escape

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

Constitutive activation of the phosphoinositide 3-kinase/AKT signaling pathway is frequently observed in high-grade gliomas with high frequency of losing PTEN tumor suppressor. To identify transcriptomic profiles associated with a hyperactivated PI3K pathway, RNA-sequencing analysis was performed in a glioblastoma cell line stably expressing PTEN. RNA-sequencing revealed enriched transcripts of pro-inflammatory mediators, and among the genes that displayed high differential expression was the secreted glycoprotein YKL-40. Treatment with chemical inhibitors that target the PI3K/AKT pathway elicited differential effects on YKL-40 expression in selected GBM cell lines, indicating that its expression displayed tumor cell-specific variations. This variability appeared to be correlated with the ability to transactivate the immune signaling molecules JAK2 and STAT3. In summary, the differential expression of the immunomodulatory molecule YKL-40 may affect the treatment efficacy of PI3K/AKT-based pathway inhibitors in glioblastoma.

Topics: Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Chitinase-3-Like Protein 1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Janus Kinase 2; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Signal Transduction; STAT3 Transcription Factor

Glioblastoma multiforme (GBM) is the most common, invasive and deadly primary type of malignant brain tumor. The Phosphatidylinositol-3-Kinase/AKT (PI3K/AKT) pathway is highly active in GBM and has been associated with increased survival and resistance to therapy. The aim of this study is to investigate the effects of AKT inhibition in combination with the current standard of care which consists of irradiation and temozolomide (TMZ) on human malignant glioma cells growing adherent and as multicellular spheroids in vitro.. The effects of the allosteric inhibitor MK2206 combined with irradiation and TMZ were assessed on glioma cells growing adherent and as multicellular 3D spheroids. The interaction was studied on proliferation, clonogenic cell survival, cell invasion, -migration and on expression of key proteins in the PI3K-AKT pathway by western blot.. A differential effect was found at low- (1 μM) and high dose (10 μM) MK2206. At 1 μM, the inhibitor reduced phosphorylation of Thr308 and Ser473 residues of AKT in both adherent cells and spheroids. Low dose MK2206 delayed spheroid growth and sensitized spheroids to both irradiation and TMZ in a synergistic way (Combination index <0.35). In contrast, neither low nor high dose MK2206 did enhance therapy sensitivity in adherent growing cells. Effective inhibition of invasion and migration was observed only at higher doses of MK2206 (>5 μM).. The data show that a 3D spheroid model show different sensitivity to irradiation when combined with AKT inhibition. Thereby we show that MK2206 has potential synergistic efficacy to the current standard of care for glioma patients.

Topics: Antineoplastic Agents, Alkylating; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Dacarbazine; Drug Synergism; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular; Temozolomide

Receptor tyrosine kinase (RTK) targeted therapy has been explored for glioblastoma treatment. However, it is unclear which RTK inhibitors are the most effective and there are no predictive biomarkers available. We recently identified the RTK AXL as a putative target for the pan-RTK inhibitors cediranib and sunitinib, which are under clinical trials for glioblastoma patients. Here, we provide evidence that AXL activity can modulate sunitinib response in glioblastoma cell lines. We found that AXL knockdown conferred lower sensitivity to sunitinib by rescuing migratory defects and inhibiting apoptosis in cells expressing high AXL basal levels. Accordingly, overactivation of AXL by its ligand GAS6 rendered AXL positive glioblastoma cells more sensitive to sunitinib. AXL knockdown induced a cellular rewiring of several growth signaling pathways through activation of RTKs, such as EGFR, as well as intracellular pathways such as MAPK and AKT. The combination of sunitinib with a specific AKT inhibitor reverted the resistance of AXL-silenced cells to sunitinib. Together, our results suggest that sunitinib inhibits AXL and AXL activation status modulates therapy response of glioblastoma cells to sunitinib. Moreover, it indicates that combining sunitinib therapy with AKT pathway inhibitors could overcome sunitinib resistance.

Topics: Angiogenesis Inhibitors; Axl Receptor Tyrosine Kinase; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; ErbB Receptors; Gene Knockdown Techniques; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Indoles; Proto-Oncogene Proteins; Pyrroles; Receptor Protein-Tyrosine Kinases; Signal Transduction; Sunitinib

The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipaseD(PLD) is a novel regulator of Akt inGBM.Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Beclin-1; Cell Line; Cell Line, Tumor; Cell Survival; Culture Media, Serum-Free; Enzyme Inhibitors; Glioblastoma; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Microscopy, Confocal; Phosphatidic Acids; Phospholipase D; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.

Topics: Analysis of Variance; Astrocytes; Cell Survival; Cell Transformation, Neoplastic; Class Ia Phosphatidylinositol 3-Kinase; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; Mutagenesis; Mutation; Plasmids; Signal Transduction

Inhibition of the survival kinase Akt can trigger apoptosis, and also has been found to activate autophagy, which may confound tumor attack. In this study, we investigated regulatory mechanisms through which apoptosis and autophagy were modulated in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt to enter clinical development. In human glioma cells, Akt inhibition by MK-2206 or siRNA-mediated attenuation strongly activated autophagy, whereas silencing of eukaryotic elongation factor-2 (eEF-2) kinase, a protein synthesis regulator, blunted this autophagic response. Suppression of MK-2206-induced autophagy by eEF-2 silencing was accompanied by a promotion of apoptotic cell death. Similarly, siRNA-mediated inhibition of eEF-2 kinase potentiated the efficacy of MK-2206 against glioma cells. Together, these results showed that blunting autophagy and augmenting apoptosis by inhibition of eEF-2 kinase could modulate the sensitivity of glioma cells to Akt inhibition. Our findings suggest that targeting eEF-2 kinase may reinforce the antitumor efficacy of Akt inhibitors such as MK-2206.

Topics: Animals; Apoptosis; Autophagy; Cell Line, Tumor; Elongation Factor 2 Kinase; Female; Glioblastoma; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Nude; Mitochondria; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Ribosomal Protein S6 Kinases; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases; Transfection; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins