PTPS-22DUAL mTOR KINASE INHIBITOR (MLN0128) MARKEDLY INDUCES GROWTH SUPPRESSION AND APOPTOSIS IN DIFFUSE INTRINSIC PONTINE GLIOMA CELL LINES

Abstract

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. The Target of Rapamycin Complex 1 and 2 (mTORC1/2) regulate protein translation, cell growth, survival, invasion and metabolism. Primary DIPG tumors harbor mutations in PTEN, AKT, and PI3K. Inhibition of TORC1 with rapalogs is minimally effective in DIPG, however, the efficacy of dual mTOR kinase inhibitors has not been examined in this tumor type. METHODS: Three DIPG cell lines (JHHDIPG1, SF7761, and SUDIPG XIII), maintained in medium with EGF and FGF, were treated for 4-72 hours with DMSO or 1-50 nM levels of the TORC1/2 kinase inhibitor MLN0128. Cell growth analysis was performed using MTS reagent and measured in 490-nm absorbance. Protein was extracted from cell lysates and Western blotting was performed using primary antibodies specific for pAKT, AKT, pS6, S6, and cleaved PARP, as well as β-actin. Immunofluorescence staining against cleaved caspase 3 (CC3) and BrdU were done to evaluate apoptosis and proliferation, respectively. RESULTS: Both pAKT and pS6 were highly expressed in these DIPGs cell lines. pAKT and pS6 were inhibited after 25nM and 10-25nM levels of MLN0128, respectively. Cell growth of DIPGs (MTS assay) was markedly inhibited by 25-50 nM MLN0128 (SF7761: 10 nM p = 0.0002, 25/50 nM p < 0.0001; SUDIPG XIII: 25 nM p = 0.0065, 50 nM p = 0.0003, all vs vehicle). Decreased proliferation (%BrdU: JHHDIPG1 p = 0.0001, SF7761 p < 0.0001, SUDIPG XIII p < 0.0003, all vehicle vs 25 nM) and increased apoptosis (%CC3: JHHDIPG1 p = 0.0004, SF7761 p = 0.0005, SUDIPG XIII p = 0.0032, all vehicle vs 25 nM; increased cleaved PARP) were seen after treatment. CONCLUSIONS: In these DIPG cell lines, the mTOR pathway is active, and inhibition of the pathway suppresses growth and induces apoptosis. These effects may be induced as a consequence of inhibition of both mTORC1 and mTORC2. Dual mTOR inhibition is a promising candidate for DIPG treatment.