| In Vitro | In vitro activity: PRC2 methyltransferase assay: Recombinant five-component PRC2 (EZH2/EED/SUZ12/RBBP4/AEBP2) was co-expressed in Sf9 and purified as described. PRC2 activity was measured using a radiometric Scintillation Proximity Assay (SPA) performed in 384-well OptiPlates (Perkin Elmer). For IC50 determination, 2.3 nM PRC2 was incubated for 90 min at RT with 1 uM histone H3-lys(biotin) (Anaspec), 1.5 uM SAM (NEB), and 500 nM 3H-SAM in 20 uL reaction buffer (50 mM Tris pH 8.5, 5 mM DTT, and 0.01% Tween-20) containing compound or DMSO. Reactions were quenched with TCA and, following the addition of PVT streptavidin-coated SPA beads (Perkin Elmer; 40 uL of 140 ng diluted in PBS), incubated for 1 hr at RT. CPM values were measured using the TopCount NXT plate reader. Percent activity values were calculated by setting the average background (no-enzyme wells) to 0% and the average DMSO wells to 100% activity. Standard deviations were determined from four replicate measurements for each compound concentration. Data were analyzed and plotted using GraphPad PRISM v6, using the ‘log(inhibitor) vs normalized response –variable slope’ analysis module to calculate IC50.
Kinase Assay: For determination of JQEZ5 mechanism of action and Ki values, reactions were carried out as described above in the presence of varying concentrations SAM/3H-SAM (at a 1:20 ratio) with a fixed concentration of 1 uM histone H3 peptide. Data were analyzed and plotted using ‘Enzyme Kinetics –inhibition’ and ‘Enzyme Kinetics – substrate versus velocity’ analysis modules in GraphPad PRISM v6
Cell Assay: H661, H292 and H522 human lung carcinoma cell lines were purchased from and authenticated through routine STR analysis and cytogenetic studies by ATCC. H969, H2250 and H2258 cell lines were a kind gift from Dr. Kenneth Huffman at UT Southwestern Medical Center; hTBE cells were a kind gift from Dr. William C. Hahn at Dana-Farber Cancer Institute. hTBE cells were received and last authenticated in 2011; all other cells were received and last authenticated by standard methods in 2014. Human lung carcinoma cell lines were cultured in RPMI-1640/10% FBS/1% penicillin–streptomycin. 293ft cells (Invitrogen) were cultured in Dulbecco's Modified Eagle Medium (DMEM)/10% FBS/1% penicillin–streptomycin. Fresh murine lung tumor nodules were minced and cultured in 100-mm dishes with RPMI-1640/10% FBS/1% penicillin–streptomycin. All cells were cultured at 37°C in a humidified incubator with 5% CO2. |
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| In Vivo | JQEZ5 was dissolved in DMSO and then diluted 1:10 in 10% (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich). DMSO (vehicle) was dissolved 1:10 in 10% (2-Hydroxypropyl)-β-cyclodextrin. Tumor-bearing GEMMs were monitored for onset of symptoms (breath distress) and then treated with JQEZ5 for three weeks (75 mg/kg IP daily). Tumors were visualized by MRI and tumor volume of the lungs was calculated using 3D Slicer. For xenograft experiments, H661 cells were dissociated into single cells, counted and resuspended at 2×106 cells per 250 μl of 1:1 media/matrigel (BD). Eight- to 12-week-old female Foxn1nu/Foxn1nu (Nude) mice (Harlan) were injected subcutaneously with 2 × 106 cells in two to three spots on the flanks. Tumors were allowed to grow to an approximate size of 200 mm3 (~10 weeks) and the mice were randomized for vehicle (n=3) or JQEZ5 administration (n=6, 75 mg/kg/d, i.p.) for 18 days. Tumor growth was measured by caliper measurements and tumor volume was calculated by standard methods. All mice were housed in pathogen-free animal facilities, and all experiments were performed with the approval of the Animal Care and Use Committee at Harvard Medical School and Dana-Farber Cancer Institute. |
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