Azacytidine

All 10 anticancer drugs were selected based on their selective function and importance as drugs that are frequently used in the clinic. Ten drug concentrations were tested for each drug. hESC cells were grown on a 96 well plates in a triplicate manner. hESC density was 15,000 cells per well. For each drug, six plates were used to allow six time points per concentration. The drug in each concentration was added on Day ‘0’ and the medium was replaced every 24 h. Cell viability was assessed by a CellTiter‐Glo luminescent cell viability assay according to the manufacturer's instructions (Promega) and cell viability was monitored following 1, 2, 3, 6, 10 and 13 days. Luminescence reads for the target genes were normalized to control conditions, and the replicate experiments were averaged. This comprehensive calibration regime allowed a careful selection of concentrations that produce significant cell death yet allow some cell recovery. All anticancer drugs in this study: Azacytidine, Bleomycin, Vorinostat, Imatinib, Sunitinib, Vemurafenib, Methotrexate, Olaparib, Ibrutinib and Enzalutamide, were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Preparations of all drugs were done according to vendor protocols.

In order to assess the resistance or insensitivity of TP53 LoF against various anticancer drugs, we applied the cell competition assay. In this assay, GFP‐labelled, TP53 KO cells were mixed in predetermined proportions with WT cells, and changes in the proportions of the labelled cells were measured by FACS analysis. The comparison of control plates (mixed cells with standard medium) versus drug‐treated plates enabled the distinction between a mutation growth advantage and response to drug treatment. TUBB::GFP TP53^−/− hESCs and WT hESCs were grown on Matrigel‐coated plates and harvested by TrypLE Select (Thermo Fisher Scientific) upon well confluency. WT and TUBB::GFP TP53^−/− cells were mixed in 98%:2% respectively, and were re‐seeded on 6‐well plates (Day −1). On Day 0, cells were harvested and prepared for FACS analysis (below). Control and experiment plates were kept with medium replacement (mTeSR, with or without anticancer drugs). The drugs tested are azacytidine, methotrexate and olaparib (Cayman Chemical, Ann Arbor, Michigan, USA) and the concentrations were as used in the genome‐wide screen. Cell competition monitoring was performed on Days 0, 5 and 9.