Drug–response assays

400 KEP1.23 or KEP2.E3 cells expressing GFP or Myc; 1,000 KEP2.E3 cells expressing GFP, Eif4b, or Melk; 700 primary control or resistant tumor cells; 4,000 MDA-MB-468 cells; or 5,000 SUM52PE, MCF7, or T47D cells were seeded per well in 96-well flat-bottomed plates. After 24 h, cells were subjected to either the mTORi AZD8055 (0.1 nM–1 µM range; AstraZeneca) or everolimus (#HY-10218; MedChem Express; 0.03–32 nM range), the PI3Kα inhibitor alpelisib (#HY-15244; MedChem Express; 80 nM–80 µM range), the pan-class I PI3Ki buparlisib (#HY-70063; MedChem Express; 10 nM–10 µM range), or the MYCi KJ-Pyr-9 (#HY-19735; MedChemExpress; 800 nM–80 µM range), each for 3 d. To measure cell viability upon combined mTOR and MYC inhibition, cells subjected to AZD8055 were additionally treated with 8 μM KJ-Pyr-9. Cell viability was assayed using Sulforhodamine B (SRB) staining. Cells were fixed with 50% ice-cold trichloroacetic acid in H₂O for 2 h at 4°C, washed five times with tap water, and air-dried. Cells were then stained for 30 min with 0.4% SRB–1% acetic acid in H₂O at room temperature and washed five times with 1% acetic acid-H₂O. Bound SRB was dissolved with 10 mM Tris-H₂O and optical density was measured at 540 nm with an Infinite 200 PRO plate reader (Tecan). For KEP2.E3 cells expressing Eif4b or Melk, cell viability was assayed using CellTiter-Blue reagent (#G808A; Promega) for 4.5 h and an Infinite M Plex plate reader (Tecan). Long-term colony formation assays were performed in 6-well plates precoated with laminin by using Rac-11P cells as previously described (Schipper et al., 2019). Cells were seeded at 5,000 cells per well and treated the following day at the indicated concentrations. After 10 d of treatment, cell viability was quantified by incubating the cells with Cell-Titer Blue reagent. Cells were then fixed with 4% formaldehyde and stained with 0.1% (wt/vol) crystal violet. Plates were imaged using GelCount (Oxford Optronix). Drug–response curves were modeled using [Inhibitor] versus response with variable slope (four parameters) and least squares regression in Prism (version 8, GraphPad Software). The Bliss independence model was applied to evaluate synergy between AZD8055 and KJ-Pyr-9 (Greco et al., 1995).