Bimiralisib

Response to single drugs or drug combinations was assessed after 72 h of exposure to increasing doses of the drug followed by an MTT assay. Copanlisib, duvelisib, umbralisib, everolimus, bimiralisib, vincristine, 5-azacitidine, masitinib, stattic and tocilizumab were purchased from Selleckchem, Lin28-1632 (LIN1632) from R&D Systems (Minneapolis, MN, USA), and human recombinant interleukin 6 (IL-6; CYT-213) from Prospec (Rehovot, Israel). Loncastuximab tesirine was kindly provided by ADC Therapeutics (Epalinges, Switzerland). Details are provided in the Online Supplementary Methods.

Between 1500 and 2000 cells per well of Plat-E, U2OS, SJSA1, HOS, and MG63 were seeded in a 384-well plate using a Multidrop Combi Reagent Dispenser (ThermoFisher Scientific) in 25 µL of media. Six thousand OHSU-SARC001 cells per well were seeded in a 96-well plate manually. Plat-E, U2OS, SJSA1, HOS, and MG63 were seeded using D10 media, whereas OHSU-SARC001 was seeded using DMEM:F12. Cells were cultured for 3–5 d depending on doubling time. Inhibitors (LY3023414, everolimus, bimiralisib, rapamycin, trametinib, doxorubicin [Selleckchem], vincristine [MedChemExpress)], actinomycin [MedChemExpress], DMSO, and staurosporine [Selleckchem]) were added using a D300 Digital Dispenser (Hewlett-Packard) ranging from 0.001 to 10 µM. Dose–response assays involving ipatasertib and afuresertib (MedChemExpress) with ranges of 0.001 to 10 µM were added manually to 96-well plates. Plat-E-, U2OS-, SJSA-, HOS-, and MG63-containing plates were incubated for 72 h at 37°C and 5% CO₂. OHSU-SARC001 cells were incubated for 6 d (given slow doubling time) at 37°C, 5% CO₂. Viability was measured using a Cell Counting Kit-8 (Bimake) and read on a Biotek Synergy 2 plate reader.