The dual mTOR kinase inhibitor TAK228 inhibits tumorigenicity and enhances radiosensitization in diffuse intrinsic pontine glioma

Abstract

Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKTand PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type. Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis. TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p=0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.

Inhibition of enhancer of zest homologue 2 is a potential therapeutic target for high‐MYC medulloblastoma

“…Cells were dissociated and seeded into 96‐well plates at a density of 2000 per well in 200 μL of medium and treated with DMSO, 150–200 nmol/L of GSK343 or 150–200 nmol/L of EPZ6438 and incubated in 5% CO 2 at 37°C for up to 120 h. For the plate readings, 20 μL of MTS solution was added to each well after 72 h post‐plating and incubated for 1 h, protected from light. The optical density at 490 nm was subsequently measured by spectrophotometer as previously described …”

“…Cells incubated with IgG1 control in parallel were used to set the gates. The percentage of Ki67 positive cells was determined using the Muse® Cell Analyzer as previously described …”

“…The optical density at 490 nm was subsequently measured by spectrophotometer as previously described. 14,15 Cell proliferation assay Cells were treated with DMSO, 100-200 nmol/L of GSK343 or 100-200 nmol/L of EPZ6438 for 72 h. Cells were subsequently fixed and stained for Ki67 per manufacturer's instructions using the Muse ® Ki67 Proliferation Assay (Millipore). Cells incubated with IgG1 control in parallel were used to set the gates.…”

“…The percentage of Ki67 positive cells was determined using the Muse ® Cell Analyzer as previously described. 14,15 Apoptosis assay DAOY-MYC and UW228-MYC cells were treated with DMSO or 200 nmol/L EPZ6438 for 72 h. Apoptosis was measured using the Muse ® Annexin V and Dead Cell Assay per the manufacturer's protocol. Cells were resuspended in DMEM/F12 without phenol red with 1% bovine serum albumin before analysis.…”

MGMT Expression Contributes to Temozolomide Resistance in H3K27M-Mutant Diffuse Midline Gliomas

“…NGT16, SF8628, U87MG, T98G cells were grown as adherent monolayer cultures in 10% FBS DMEM. SF7761, JHH-DIPG1 cells were grown as sphere cultures in EF20 medium composed with Neurobasal Medium (Thermo), 20 ng/ml EGF (Peprotech, Rocky Hill, NJ, USA), 20 ng/ml FGF (Peprotech), 2% B27 supplement (Thermo), 0.25% N2 supplement (Thermo), 3 mM L-Glutamine (Thermo), 2 µg/ml Heparin (Sigma), and 1% Antibiotic-Antimycotic (Thermo) (30,31).…”