1. Volume 19, Issue 13, Pages 2665-2680 (June 2017)
Purine Nucleotide Availability Regulates mTORC1 Activity through the Rheb GTPase 
Natasha Emmanuel, Shoba Ragunathan, Qin Shan, Fang Wang, Andreas Giannakou, Nanni Huser, Guixian Jin, Jeremy Myers, Robert T. Abraham, Keziban Unsal-Kacmaz 
Cell Reports 
Volume 19, Issue 13, Pages (June 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 2665-2680DOI: (10.1016/j.celrep.2017.05.043)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1
Differential Effects of AG2037 and PTX on mTORC1 Signaling and Cell Cycle Progression
(A) NSCLC cells lines were treated overnight with vehicle (DMSO), 1 μM AG2037, or 1 μM PTX. The table indicates drug IC50 values derived from sulforhodamine B (SRB) cell proliferation assays.
(B) H460 cells were treated overnight with the indicated concentrations of AG2037 and PTX.
(C) Cell cycle distributions were determined by flow cytometric analyses of H460 cells treated with DMSO, 100 nM AG2037, or 500 nM PTX for the indicated times. The table depicts the percentages of cells in G1, S, and G2/M phases at each time point.
(D) A549 cells were treated for 24 hr with 150 nM AG207 and 500 nM PTX in the presence of medium (M), medium supplemented with 32 μM hypoxanthine (H), 5.6 μM thymidine (T), or both (H+T).
(E) A549 cells, stably transduced with doxycycline-inducible shRNAs targeting GARFT, were treated with 0.25 μM doxycycline for 2 days.
(F) Cells were treated as in (E) in the presence of 32 μM hypoxanthine.
See also Figures S1 and S2.
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4. Figure 2 Rescue Experiments with Exogenously Added Purine Bases
(A) Schematic of the purine biosynthesis and salvage pathways.
(B) Cells were treated overnight with 150 nM of AG2037 or 500 nM PTX in the culture medium or medium supplemented with 30 μM adenine (A) or guanine (G).
(C and D) A549 (C) and A427 (D) cells were treated for 4 days with the indicated drug concentrations, with or without adenine and guanine as in (B), and cell proliferation assays were performed. Data are presented as mean ± SD from triplicate samples.
(E) A549 cells were treated overnight with 150 nM AG2037, 500 nM AVN-944, or 1 μM MPA.
(F) Cells were treated overnight with 500 nM AVN-944, in medium only or in medium supplemented with adenine or guanine.
(G) Cells were treated for 4 days with the indicated drug concentrations with or without adenine and guanine. Data are presented as mean ± SD from triplicate samples.
(H) A549 cells were treated overnight with both 150 nM AG2037 and 500 nM AVN-944 in the absence or presence of adenine (Ade).
(I) Schematic depicting the distinct steps in purine biosynthesis targeted by AG2037 versus AVN-944.
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5. Figure 3 Metabolic Profiling of AG2037- and AVN-944-Treated Cells
(A) Schematic of the relevant steps in de novo purine biosynthesis affected by AG2037 and AVN-944 (left). GAR and IMP metabolites were measured by liquid chromatography-mass spectrometry (LC-MS) in A427 cells treated with 150 nM AG2037 and 500 nM AVN-944 for the time periods indicated (right). Error bars represent SEM from three independent samples per condition.
(B) Heatmap showing log2 fold changes of metabolites in A427 cells treated with AG2037 or AVN-944 for the indicated time periods. Data are represented as mean fold change relative to vehicle controls from three independent samples. The table depicts p values of 24-hr drug treatment data compared with a time-matched DMSO control. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < (two-tailed t test).
(C) Metabolite levels in A427 cells treated for 24 hr with AG2037 and AVN-944 in medium supplemented with adenine or guanine. LC-MS data are represented as mean fold change relative to control ± SEM from three independent samples. ∗p < 0.05, ∗∗p < (two-tailed t test).
See also Figure S3.
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6. Figure 4
Inhibition of De Novo Purine Biosynthesis Suppresses mTORC1-Stimulated Pyrimidine Biosynthesis and Translation Initiation
(A) Pyrimidine metabolites were measured by LC-MS in A427 cells treated with 150 nM AG2037 for the indicated time periods. Data are represented as mean-fold change relative to control, ± SEM from three independent samples. ∗p < 0.05, ∗∗p < (two-tailed t test).
(B) N-carbamoyl-L-aspartate was measured in A427 cells treated for 24 hr with 150 nM AG2037 in the presence of adenine or guanine. Data are represented as mean fold change compared with vehicle-treated controls ± SEM from three independent samples per condition. ∗p < 0.005, ∗∗p < (two-tailed t test).
(C) A427 cells were treated overnight with 150 nM AG2037 in medium with or without Ade.
(D) A427 cells were treated overnight with 150 nM AG2037, 30 nM CCI-779, or 150 nM WYE (WYE-132). Extracts were precipitated with 7-methylguanosine 5′-triphosphate (m7GTP)-conjugated beads.
(E) Mice bearing established (200-mm3 volume) A427 xenografts were randomized and treated with 10 mg/kg or 20 mg/kg AG2037 or drug vehicle only. Data are presented as mean tumor volume ± SEM (n = 10 mice/data point). ∗∗∗∗p < (two-tailed t -test).
(F) Mice bearing established tumors (300- to 400-mm3 volume) were treated with 20 mg/kg AG2037 on days 1, 4, and 7. On day 8, tumor tissues were harvested for immunoblotting.
See also Figure S4.
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7. Figure 5
Effect of AG2037 Exposure on RhebGTP Levels and Rheb Posttranslational Processing
(A) HEK293T cells transfected with the indicated expression plasmids were treated overnight with 300 nM AG2037. Cells were deprived of amino acids (AAs) for 2 hr and stimulated for 30 min with a 2-fold concentration of AAs. Cellular extracts were immunoblotted for the indicated proteins.
(B) HEK293T cells were transiently transfected with wild-type Rheb and treated overnight with 1 μM AG2037 in the presence or absence of 30 μM Ade or 30 μM guanine (Gua). Extracts were immunoprecipitated with RhebGTP-specific antibody. Samples incubated with a non-hydrolyzable GTP analog (GTPγS) or GDP were used as positive and negative controls, respectively. The graph depicts densitometric quantification of Rheb-GTP relative to total Rheb from four independent experiments ± SD. ∗p < 0.05, ∗∗∗p < (two-tailed t test).
(C) HEK293T cells transiently transfected with a Myc-tagged wild-type Rheb construct were treated overnight with 1 μM AG2037 or 10 μM FTI-277.
(D–F) Immunoprecipitates from (C) were analyzed by MS to determine the total levels of Rheb (D), non-farnesylated Rheb (SSC-carboxyaminomethyl-SVM) (E), and farnesylated Rheb (SSC-farnesyl) (F). The data in (E) and (F) were normalized to the total level of Rheb protein ± SD from duplicates.
(G) HEK293T cells were transiently transfected with wild-type, C181S, or S20N mutant Rheb. Cells expressing wild-type Rheb were treated overnight with vehicle or 1 μM AG2037.
(H) HEK293T cells were transiently transfected with wild-type or C181S Rheb. The mutant-transfected cells were treated overnight with vehicle or 1 μM AG2037. Extracts were immunoprecipitated with RhebGTP-specific antibody, followed by anti-Rheb immunoblotting. The graph depicts densitometric quantification of Rheb-GTP relative to total Rheb from three independent experiments ± SEM. ∗, overexpressed Rheb; ∗∗, endogenous Rheb.
See also Figures S5–S7.
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8. Figure 6 Attenuation of AG2037 Activity in TSC1-Null MEFs
(A) TSC1+/+ and TSC1−/− MEFs were serum-deprived overnight and stimulated for 30 min with 100 nM insulin.
(B) Cells were starved overnight with concurrent AG2037 treatment and stimulated with insulin as in (A) or further starved of AAs by replacing the medium with Hank’s balanced salt solution (HBSS+) for 2 hr and stimulated for 30 min with a 2-fold concentration of AA.
(C) Cells were treated overnight with 750 nM AG2037, 600 nM AVN-944, or 6 nM CCI-779.
(D) Cells were treated overnight with increasing AG2037 concentrations (TSC1−/−, 0.1–10 μM; TSC1+/+, 0.03–3 μM).
(E) Cells were treated for 4 days with the indicated concentrations of AG2037, and cell viability was assessed. Data are represented relative to vehicle controls and with three biological replicates ± SD.
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9. Figure 7 AG2037 Disrupts the Lysosomal Localization of Rheb and TSC2
(A) HeLa cells were serum-starved overnight in the presence of 500 nM AG2037 and stimulated for 30 min with insulin.
(B) Cells were treated as in (A), immunostained for TSC2 (green) and LAMP1 (red), and imaged by confocal microscopy. Scale bars, 20 μm.
(C) Percent colocalization and Pearson’s correlation coefficient (PCC) are graphed as mean ± SEM.
(D) Cells treated as in (A) were immunostained for mTOR (green) and LAMP1 (red).
(E) Confocal images from (D) were quantified as in (C). Scale bars, 20 μm.
(F) Cells were treated as in (A), and lysosomes were purified by differential centrifugation.
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