Proteotoxicity response genes whose translation is inhibited in mutant eIF-4E-containing MM cells.

<p>List of genes involved in proteotoxic responses with significant decreases in translational efficiency.</p

Effect of CGP57380.

<p><b>A</b>) 8226 (left panel) or ANBL-6 (right panel) MM cells pre-treated with CGP57380 for 30 mins at varying concentrations, followed by addition of IL-6 (100 U/ml) for 3 hrs or no IL-6 and then immunoblot assay performed. <b>B</b>) 8226 (white bars) and ANBL-6 (black bars) seeded at 3×10⁵ cells/ml and treated +/− CGP57380 (at concentrations shown in uM) +/− IL-6 (100 U/ml) for 72 hrs. Viable cell counts enumerated by trypan blue exclusion. Results shown are means+/−SD, (n = 3). <b>*</b> denotes significant (p<0.05) inhibition of cell numbers versus control (no CGP57380). <b>C</b>) Two primary MM specimens cultured +/− CGP57380 for 72 hrs with viable cell recovery enumerated (cell counts presented as mean+/−SD of triplicate samples). In one patient (black bars) cells also exposed +/− IL-6 (100 U/ml) and in a second patient (white bars) no IL-6 was used (insufficient cell numbers harvested for these extra groups). <b>D</b>) 8226 or ANBL-6 cells pre-treated with cercosporamide (cer) for 30 mins at varying concentrations, followed by the addition of IL-6 for 3 hrs or no IL-6 and then immunoblot assay performed. E) 8226 and ANBL-6 cells seeded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094011#pone-0094011-g002" target="_blank">fig 2B</a> and treated +/− cercosporamide at varying concentrations +/− IL-6 for 72 hrs. Viable cell counts are means+/−SD (n = 3) and * denotes significant (p<0.05) inhibition versus control.</p

Effects on global translation and lambda light chain synthesis.

<p><b>A</b>) AHA incorporation is tested in MM cells stimulated with IL-6 for 30, 60, 120 or 180 mins. In <b>B</b>), AHA incorporation is tested in IL-6-stimulated EV-, WT or mutant SA eIF-4E-transfected MM cells. In <b>C</b>), EV or SA mutant-expressing cells are pre-treated +/− pp242, followed by IL-6. Bar graphs under panels are means+/−SD of 4 separate experiments. * denotes statistically significant increase in AHA incorporation induced by IL-6 in A and B and decrease versus IL-6-stimulated control in C). <b>D</b>) Lambda light chain ELISA assay in MM cell lysates, comparing EV- vs mutant eIF-4E-transfected cells +/− IL-6 and +/− pp242. Data are means+/−SD, n = 4. * denotes statistically significant difference from corresponding controls.</p

Ribosome profiling of EV vs mutant eIF-4E-transfected MM cells.

<p><b>A</b>) Different categories of 166 genes demonstrating significantly inhibited translation in mutant-expressing cells. <b>B</b>) Immunoblot analysis of protein expression in EV-, WT and mutant (MU)-expressing MM cells. <b>C</b>) Immunoblot analysis of protein expression in EV vs mutant (MU) eIF-4E-expressing MM cells +/− IL-6 for 48 hrs.</p

Activation of MNK kinases.

<p><b>A</b>) MM lines exposed to IL-6 (100 U/ml) for 3 hrs followed by immunoblot assay for phospho-MNK and total MNK expression. Fold increase is determined by densitometric ratio of MNK-P/MNK-total and represents the mean of 3 independent experiments. <b>*</b> denotes IL-6-induced increase is statistically significant (p<0.05). <b>B</b>) ANBL-6 cells exposed to IL-6 for 0, 60, 120 or 180 mins, followed by immunoblot assay. <b>C</b>) Primary cells from 4 patients exposed to IL-6 or media (C) followed by immunoblot assay. <b>D</b>) ANBL-6 cells treated with IL-6 for increasing durations, followed by immunoprecipitation with anti-MNK1, MNK2 or control IgG. Immunoprecipitates tested for phosphorylation of eIF-4E in vitro.</p

Metabolism genes whose translation is inhibited in mutant eIF-4E-containing MM cells.

<p>List of all genes related to metabolism with significant decreases in translational efficiency. ‘RNA” value given is log₂ change in RNA abundance in mutant-expressing cells vs EV-cells.</p

Specific blockade of Rictor-mTOR association inhibits mTORC2 activity and is cytotoxic in glioblastoma

<div><p>A small molecule which specifically blocks the interaction of Rictor and mTOR was identified utilizing a high-throughput yeast two-hybrid screen and evaluated as a potential inhibitor of mTORC2 activity in glioblastoma multiforme (GBM). <i>In vitro</i>, CID613034 inhibited mTORC2 kinase activity at submicromolar concentrations and in cellular assays specifically inhibited phosphorylation of mTORC2 substrates, including AKT (Ser-473), NDRG1 (Thr-346) and PKC<b>α</b> (Ser-657), while having no appreciable effects on the phosphorylation status of the mTORC1 substrate S6K (Thr-389) or mTORC1-dependent negative feedback loops. CID613034 demonstrated significant inhibitory effects on cell growth, motility and invasiveness in GBM cell lines and sensitivity correlated with relative Rictor or SIN1 expression. Structure-activity relationship analyses afforded an inhibitor, JR-AB2-011, with improved anti-GBM properties and blocked mTORC2 signaling and Rictor association with mTOR at lower effective concentrations. In GBM xenograft studies, JR-AB2-011 demonstrated significant anti-tumor properties. These data support mTORC2 as a viable therapeutic target in GBM and suggest that targeting protein-protein interactions critical for mTORC2 function is an effective strategy to achieve therapeutic responses.</p></div

Effects of JR-AB2-011 treatment on GBM tumor growth in mice.

<p><b>(</b>A) Tumor burden of SCID mice implanted with LN229 cells and treated with the indicated schedules of vehicle, JR-AB2-011 (4 mg/kg/d) and JR-AB2-011 (20 mg/kg/d) for ten consecutive days and tumor growth assessed every two days following initiation of treatment (start, day 0). *, <i>P</i> < 0.05, significantly different from vehicle, JR-AB2-011 (4 mg/kg/d) and JR-AB2-011 (20 mg/kg/d). (B) Overall survival of subcutaneous LN229 tumors receiving the indicated treatment schedules. (C) Ki-67 positive cells were identified via immunohistochemical staining of sections prepared from harvested tumors at day 12 following initiation of treatment regimens (upper left panel). Apoptotic cells were identified by TUNEL assays of sections prepared from harvested tumors at day 12 following initiation of treatment regimens (upper right panel). Data are expressed as the number of positive apoptotic bodies divided by high power field (hpf; 10–12 hpf/tumor). Values are means +S.D., *, <i>P</i> < 0.05. Phospho-S⁴⁷³-AKT/total AKT protein ratio levels in tumors (lower left panel). Values are means ±S.D., *, <i>P</i> < 0.05, significantly different from vehicle, JR-AB2-011 (4 mg/kg/d) and JR-AB2-011 (20 mg/kg/d). Phospho-T³⁸⁹-S6K/total S6K protein ratio levels in tumors (lower right panel). Values are means ±S.D., *, <i>P</i> < 0.05, significantly different from vehicle, JR-AB2-011 (4 mg/kg/d) and JR-AB2-011 (20 mg/kg/d). Protein levels were quantified by Western analyses of harvested tumors from mice with the corresponding treatments as indicated and described in Material and methods.</p

Synthesis of JR-AB2-000 (CID613034) analogs.

<p>Thirty analogs were synthesized with the indicated group modification shown and further detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176599#pone.0176599.t001" target="_blank">Table 1</a>. Analog identification numbers are shown below the structures corresponding to analogs where a particular functional group is modified. Detailed synthetic procedures and NMR spectra are described in supplementary Materials and methods.</p

Summary of anti-GBM effects of CID613034 analogs.

<p>Summary of anti-GBM effects of CID613034 analogs.</p