Data shown are mean SD from n = 10 mice in each group

Data shown are mean SD from n = 10 mice in each group. tumor, represents one of the most aggressive cancers (Omuro and DeAngelis, 2013). Although signaling from PI3K and AKT to mTOR is commonly dysregulated in GBM (Brennan et al., 2013), blockade of these upstream targets minimally affects mTOR activity in glioma (Fan et al., 2009). Direct targeting using allosteric inhibitors incompletely blocks mTORC1 activity (Feldman et al., 2009; Garcia-Martinez et al., 2009; Thoreen et al., NCT-503 2009), while mTOR kinase inhibitors (TORKi) have not yet been fully evaluated in GBM. mTOR exists in two distinct complexes, mTORC1 and mTORC2 (Loewith et al., 2002). With IC50 for mTORC1 inhibition in the high picomolar range, clinically approved first generation mTOR inhibitors rapamycin and rapalogs sensitively and specifically inhibit mTORC1 through binding to the FK506 rapamycin binding (FRB) domain of mTOR with the aid of FK506 Binding Protein 12 (FKBP12) (Chiu et al., 1994; Loewith et al., 2002). Importantly, the FRB domain of mTOR is exposed in the mTORC1 but not the mTORC2 complex, which confers the mTORC1 specificity of rapalogs (Gaubitz et al., 2015). Second-generation TORKi act through orthosteric interactions with the ATP binding pocket of mTOR kinase (Feldman et al., 2009; Garcia-Martinez et al., 2009; Thoreen et al., 2009). As a result, TORKi block activation of substrates of mTORC1 and mTORC2, whereas rapalogs only impact mTORC1. (Feldman et al., 2009; Garcia-Martinez et al., 2009; Hsieh et al., Fndc4 2012; Thoreen et al., 2009). Recently developed mTORC1-directed inhibitors combine the high affinity of rapamycin for mTORC1 with the effective kinase inhibition of the TORKi MLN0128 (Rodrik-Outmezguine et al., 2016). The linker portion of this third generation mTOR inhibitor lies in a channel in the mTORC1 complex, in a manner that does not disrupt linked rapamycin binding to FKBP12 or the FRB domain of mTOR. These inhibitors thus leverage the high selectivity and affinity of rapamycin for mTORC1 to specifically deliver MLN0128 to the ATP-site of mTOR mainly in the mTORC1 complex. RESULTS mTOR is a central therapeutic target in GBM To clarify the importance of mTOR as a target in GBM, we assessed proliferation (Figure 1A), cell cycle (Figure 1B), PIP3 levels (Figure 1C), and activation of AKT, RPS6, and 4EBP1 (Figures 1D and S1) following treatment NCT-503 of LN229 cells with inhibitors targeting individual class I PI3Ks, a pan-inhibitor of class I PI3Ks, an inhibitor of AKT, an inhibitor of mTORC1, a TORKi, and a dual inhibitor of PI3K and mTOR. Decreased proliferation (Figure 1A) and arrest in G0/G1 (Figure 1B) correlated with blockade of mTORC1, assessed by decreased p-RPS6S235/236 and p-4EBP1T37/46 (Figure 1D and S1). No correlation to proliferation was evident with the abundance of PIP3 or mTORC2 inhibition, as assessed by p-AKTS473 (Figures 1C and ?and1D).1D). Only the abundance of the mTOR target p-4EBP1T37/46 correlated consistently and directly with proliferation in GBM cells (Figures 1A, ?,1D,1D, and S1). Open in a separate window Figure 1 mTOR is an attractive therapeutic target in GBM(A) LN229 cells were treated for 3 days with inhibitors against PI3K (, , , or ), pan-class I PI3K, AKT1/2, mTORC1, mTOR, and dual PI3K/mTOR inhibitors at doses indicated. Proliferation was measured by WST-1 assay. Data shown represent mean SD of triplicate measurements (Percentage growth relative to DMSO-treated control). (B) Flow cytometric analysis of cells treated as in (A) for 24 hr. Percentage of cells in G0/G1, S, and G2/M phases of cell cycle and apoptotic SubG1 fractions are indicated. Data shown represent mean SD of triplicate measurements. (C) Cells were treated as in (A) for 3 hr. Lipids were extracted and analyzed by ELISA. Data shown represent mean SD of triplicate measurements. Samples were normalized to DMSO treatment. (D) Western blotting analysis of cells from (B). Cells NCT-503 were harvested, lysed, and analyzed as indicated. Cell lysates were from a single experiment. Gels were run for the same period of time, and blots were processed with equivalent exposure times, to assure reproducibility. Representative blots from two independent experiments are shown. The names of the inhibitors against the targets shown in (A) are indicated below the.