Amg232

(±)-Nutlin-3 (Cayman Chemicals, Ann Arbor, MI, USA) was dissolved in DMSO to make 50 mM stock solution. MI-773 (MedChem Express Co. Ltd., China) and AMG232 (MedChem Express) were dissolved in DMSO to make 10 mM stock solutions. Overnight yeast cultures were washed and then diluted to OD₆₀₀ = 0.2 in 1 mL SD-Leu-Trp-His-Ade medium containing the desired concentration of the drug. The cultures were then placed in a 30 °C incubator with shaking for 3 days. Growth of the cultures was monitored by measuring the absorbance at 600 nm using the Implen Nanophotometer. This assay was also performed in a 96-well plate format with 200 μL SD-Leu-Trp-His-Ade medium containing the desired concentration of the drug in duplicate. OD₆₀₀ at different time points was measured using a Gen 5^TM (BIO-TEK Instrument, Vermont, USA) microplate reader. The average of the two OD₆₀₀ readings was calculated and used in the analysis.

The following compounds were purchased from (MedChemExpress Monmouth Junction, NJ, USA): CCT251545, MTX‐211, CUDC305, CUDC427, AMG232, ML329 and fasudil. Both 666‐15 and GSK‐J4 were purchased from R&D Systems (Minneapolis, MN, USA). Larotrectinib was purchased from Abmole (Houston, TX, USA). All other agents were purchased from Selleckchem (Houston, TX, USA). All agents were received as a dried powder and were reconstituted in DMSO (D8418, Sigma Aldrich, St. Louis, MO, USA) to a concentration of 10 mM or lower based on solubility specifications per manufacturer. Agents were plated at four concentrations (2, 0.2, 0.02 and 0.002 μM) in triplicate in a 384‐well format using the HP Tecan D300e and Perkin Elmer SciClone G3 digital dispensers. Cells were grown in T‐75 flasks until 80% confluent, trypsinised and plated at a density of 2000–2500 cells per well and incubated at 37°C with humidified 5% CO₂ for 72 h. Cell viability was measured using CellTiter‐Glo 2.0^® (G9243, Promega, Madison, WI, USA) per manufacturer's instructions. Luminescence was measured using a BioTek Synergy HT plate reader (BioTek, Winooski, VT). IC₅₀ values were determined using a nonlinear best fit method.

CellTracker™ CMFDA (5-chloromethylfluorescein diacetate) green fluorescent dye (5 µM) or CellTracker™ CMAC (7-amino-4-chloromethylcoumarin) blue fluorescent dye (10 µM) were used to stain and detect living tumor cells or T-cells. Before co-culture with T-cells, culture media was removed and pre-warmed CellTracker™ in “working solution” was added as instructed in the manufacturer’s protocol (Invitrogen, Waltham, MA). The working solution with the CellTracker™ was replaced with fresh media after 30 min of incubation at 37 °C. Green fluorescent tumor cells with or without 1 µM AMG-232 (MedChem Express) pretreatment were co-cultured with or without 25 µg/mL pembrolizumab pre-treated cytotoxic T-cells (TALL-104) with a 2:1 effector-to target cell ratio (E:T) for 12 or 16 h. RPMI-1640 media containing 20% FBS and 100 units/mL IL-2 was used in the co-culture system. For analysis of cell death, 1-µM red fluorescent ethidium homodimer-1 (EthD-1) was added and incubated for another 30 min to detect dead cells (Invitrogen, Waltham, MA). For the quantification of dead/live cells, fluorescence microscopy was used to take images at 10× magnification. The number of red/green/blue colored cells in random fields was counted by two independent investigators and expressed as a dead/live cell ratio. At least 100 cells were evaluated per sample, with three independent replicates.