Fig. 3: p53-Y220C–PLK1 bifunctional molecules selectively inhibit proliferation of TP53Y220C-mutant cells.
From: Mutant p53 protein accumulation is selectively targetable by proximity-inducing drugs
a, Structure of p53-Y220C–PLK1 bifunctional compound (PMV6-PEG4-BI2536, p53-01) used in experiments. b, Nanoluciferase signal 1 day after dose titration of p53-01 in 293T cells cotransfected with NLS–LgBiT–p53-Y220C (DBD) and mEGFP–PLK1–SmBiT (n = 6 technical replicates per condition). c, Live-cell imaging of 293T cells cotransfected with Halo–p53-Y220C ΔTAD–mCherry and mEGFP–PLK1–SmBiT, treated with p53-01. The white arrow indicates the extranuclear region containing PLK1; the red arrow indicates the nucleus. Dividing and nondividing cells are shown. mCherry (p53) and EGFP (PLK1) channels are shown separately. Two replicates of this experiment were performed; a representative replicate is shown. d, Competition of 293T cells stably expressing Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells in the presence of various compounds (PMV6, KG5-PEG4-BI2536, selinexor, BI2536, AP1867-C8-BI2536 and p53-01). Right, competition of 293T cells stably expressing Halo–p53-R273H (FL)–mCherry versus parental 293T cells in the presence of p53-01. The mCherry+ percentage on day 8 of competition normalized to that of DMSO-treated cells is shown. Two replicates of this experiment were performed; a representative replicate is shown. e, Crystal violet staining of 293T cells stably expressing Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells treated with p53-01, analyzed after 5 days. The experiment was performed in triplicate; a representative replicate is shown. f, Caspase 3/7 glo of Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells treated with p53-01 after 1 day (n = 4 technical replicates per condition). g, Proportion of early apoptotic cells (annexin+, PI−) following 1 day of treatment with p53-01, BI2536 or PMV6 at the indicated concentrations in Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells. NS, not significant. h, Competition of 293T cells stably expressing Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells in the presence of p53-01 (250 nM) alone or in combination with PMV6 (2.5 μM). i, DNA content (assessed by flow cytometry of DAPI-stained cells) following treatment of palbociclib-synchronized Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells with p53-01, BI2536 or PMV6 at the indicated concentrations for 1 day. The experiment was performed in triplicate; one representative replicate is shown. j, Western blot for PLK1 targets including cyclin B1 and phospho-PLK-binding motif of double-thymidine-block-synchronized Halo–p53-Y220C ΔTAD–mCherry versus parental 293T cells treated with p53-01, BI2536 or PMV6 at the indicated concentrations for 8.5 h. Two replicates of this experiment were performed; a representative replicate is shown.
