Allele-specific p53 mutant reactivation

Abstract

Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the National Cancer Institute's anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53(R175) mutant. Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53(R175) mutant. This compound kills p53(R172H) knockin mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele-specific mutant p53-dependent manner. This activity depends upon the zinc ion chelating properties of the compound as well as redox changes. These data identify NSC319726 as a p53(R175) mutant reactivator and as a lead compound for p53-targeted drug development.

Figure 1. Identification of thiosemicarbazones with activity in cell lines expressing mutant p53

(A) Flow-diagram of the in silico screen methodology. (B) IC50 distribution and chemical structure of the thiosemicarbazones aligning lower IC50s to higher IC50s in cells with mutant p53 (red) and WT p53 (blue), analyzed with the NCI60 dataset. In the NCI60 dataset, the human tumor cell lines are treated with 5 serial dilutions of each compound for 48 hours at 37°C. (C) Validation of the sensitivity of the compounds in the cell lines expressing mutant p53. Cell growth inhibition assays using a mouse embryonic fibroblast (MEF) cell line system consisting of (10)3 (p53^−/−), (10)3/175, (10)3/248, (10)3/273 and 3T3 (p53^+/+). Left panel: Growth inhibition with NSC319726. Right panel: Growth inhibition with NSC319725. (D) Sensitivity to NSC319726 in a set of isogenic MEF cell lines from p53^+/+, p53^−/− and p53^R172H/R172H mice. (E) NSC319725 and NSC319726 do not inhibit cell growth of WI38 (p53^+/+) human fibroblasts. (F) The sensitivity of NSC319726 in human tumor cell lines with hotspot p53 mutations. Cell growth inhibition was analyzed by MTS assay (C) or by Guava ViaCount assay (D, E, F). In both the MTS and Guava ViaCount assays, cells were treated with five serial dilutions of the compounds (0.00001 µM to 10 µM) for 3 days. The error bars are +/− SD. See also Figure S1.

Figure 2. The p53-175 mutant-dependent apoptosis induced by NSC319276

The apoptosis is measured with Annexin-V staining using Guava Nexin reagent and Guava PCA instrument. (A) Apoptosis is measured in the (10)3-derived MEF cells. (B) Apoptosis is measured in a panel of ovarian cancer cell lines, TOV112D (p53^R175H), OVCAR3 (p53^R248W) and SKOV3 (p53^−/−). The treatment is 1 µM NSC319726 (as in the other figures unless described differently) for 24 hours. (C) Growth inhibition by NSC319726 in TOV112D cells with siRNA knockdown of p53^R175H mutant protein, measured as in Figure 1E.

Figure 3. A “WT-like” conformational change in the p53-175 mutant protein induced by NSC319726

(A) Immunoflourescence of TOV112D (p53^R175H) cells using p53 conformation specific antibodies (PAB1620 for WT conformation and PAB240 for mutant conformation). The H460 cell line is used as a control to show the WT p53 conformation is not changed by NSC319726. All scale bars represent a size of 25 µm. (B) Quantification of PAB240 and PAB1620 staining indicates significant differences upon treatment with NSC319726 (p<0.0001) (t-test). The error bars are +/− SD. (C) Immunoprecipitation of mutant p53 protein in TOV112D cells using PAB240 and detected by p53 (FL393) by Western blot. (D) Immunoflourescence of MEF p53^R172H/H cells using the PAB240 mutant specific antibody. In all experiments (A, C, D) cells are treated with NSC319726 (1 µM) for 6 hours before harvesting for immunostaining or immunoprecipitation.

Figure 4. Restoration of site-specific p53-175 mutant protein transactivational function by NSC319726

(A) TOV112D and SKOV3 cells are treated with NSC319726 or etoposide and protein extracts are analyzed by Western blot with antibodies directed against p21, p53. GAPDH is used as the internal (loading) control. (B) Stability of p53 mutant protein in TOV112D (p53^R175), OVCAR3 (p53^R248), and SW620 (p53^R273) cells treated with NSC319726 (1 µM) with/without Nutlin-3 (5 µM) over a 24-hour time course. Protein levels are shown by Western blot using antibodies to p53 (DO-1) or either actin or GAPDH. (C) Chromatin Immunoprecipitation (ChIP) assay. Lysates from TOV112D cells treated with NSC319726 (1 µM) and HCT116 cells treated with etoposide (20 µM) for 6 hours are subject to immunoprecipitation using anti-p53 antibody (DO-1) followed by PCR of p53 recognition elements (p53REs) of p21, PUMA and MDM2. HCT116 treated with etoposide is used as a positive control for functional p53 DNA-binding property. ChIP with anti-RNA polymerase II antibody and PCR for GAPDH is also used as a positive control. (D) qRT-PCR of p21, MDM2 and PUMA in TOV112D, OVCAR3 and SKOV3 cells with treatment of NSC319726 (1 µM) for 24 hours. The RNA is extracted from the cells using Qiagen Rneasy kit and the gene expression level is measured by quantitative RT-PCR using TaqMan gene expression assays. (E) TOV112D cells are transfected with a plasmid bearing the 20-bp p53 response element (p53RE) in p21 promoter region, and then treated with NSC319726 (1 µM) for 24 hours, followed by luciferase reporter assay. The error bars are +/− SD. (F) Microarray analysis of NSC319726 (1 µM, 24 hours) treated and control TOV112D cells comparing gene expression levels of p53 targets. The list of the genes is presented in Table S1. The heatmap is shown with the color scale indicating fold change. The data from the microarrays have been deposited in the GEO database with accession number GSE35972.

Figure 5. In vivo evidence of NSC319726 mediated p53-175 mutant reactivation

(A) Toxicity assays of NSC319726 in p53^R172H/R172H, p53^+/+ and p53^−/− mice. Mice in the top panel are administered 10mg/kg by daily intraperitoneal (IP) injection for up to 7 days. Mice in the bottom panel are administered 5 mg/kg daily (IP) for up to 7 days. (B) Immunohistochemical staining with cleaved caspase-3 antibody of spleen and thymic tissues in p53^+/+ and p53^R172H/R172H mice. All scale bars represent a size of 100 µm. (C) qRT-PCR of mRNA levels of several p53-regulated genes in p53^+/+ and p53^R172H/R172H mouse tissues. The inductions of over 1.7× are highlighted in bold. (D) Efficacy assays of NSC319726. Xenograft tumors are generated using H460, MDAMB468 and TOV112D cells and allowed to grow to 60 mm³ prior to daily intravenous administration of NSC319726 at 1 mg/kg or 0.1 mg/kg (TOV112D only). Tumor measurements are mean ± SD. The following number of animals were used, H460 (Control-5, Treatment-5), MDAMB468 (Control-9, Treatment-8), TOV112D (Control-7, Treatment 1mg/kg-7, 0.1 mg/kg-7). CTL is Control. The error bars are +/− SD.

Figure 6. Zinc ion chelation and redox changes are important for the NSC319726 mediated p53-175 mutant reactivation

(A) TOV112D cells are treated with NSC319726 with or without the presence of various concentrations of FeSO₄, followed by measurement of growth inhibition. The NSC319726 activity is shown as 1/IC50 for cell growth inhibition. (B) TOV112D cells are treated with NSC319726 with or without addition of various concentrations of ZnCl₂, followed by measurement of growth inhibition. The NSC319726 activity is shown as 1/IC50 for cell growth inhibition. (C) Ratio of reductant GSH and oxidative GSSG in the p53^R175H cells upon NSC319726 (1 µM) treatment is measured at several time points (p=0.0057 at 1hr, p=0.0027 at 3hrs, and p=0.001 at 24hrs, t test). The error bars are +/− SD. (D) TOV112D cells are treated with NSC319726 using six serial dilutions (0.001 µM to 2 µM) with either N-acetyl cysteine (NAC, 5 mM) or diamide (100µM) for three days. The growth inhibition was analyzed by MTS assay as in Figure 1C. The error bars are +/− SD. See also Figure S2.