Acetylation Targeting Chimera Enables Acetylation of the Tumor Suppressor p53

Abstract

With advances in chemically induced proximity technologies, heterobifunctional modalities such as proteolysis targeting chimeras (PROTACs) have been successfully advanced to clinics for treating cancer. However, pharmacologic activation of tumor-suppressor proteins for cancer treatment remains a major challenge. Here, we present a novel Acetylation Targeting Chimera (AceTAC) strategy to acetylate the p53 tumor suppressor protein. We discovered and characterized the first p53Y220C AceTAC, MS78, which recruits histone acetyltransferase p300/CBP to acetylate the p53Y220C mutant. MS78 effectively acetylated p53Y220C lysine 382 (K382) in a concentration-, time-, and p300-dependent manner and suppressed proliferation and clonogenicity of cancer cells harboring the p53Y220C mutation with little toxicity in cancer cells with wild-type p53. RNA-seq studies revealed novel p53Y220C-dependent upregulation of TRAIL apoptotic genes and downregulation of DNA damage response pathways upon acetylation induced by MS78. Altogether, the AceTAC strategy could provide a generalizable platform for targeting proteins, such as tumor suppressors, via acetylation.

Figure 1.

Design and testing of initial p53Y220C-targeting AceTACs. (A) Crystal structure of the p53Y220C-PK9323 complex (PDB ID: 6SI0). The cross-section of the occupied binding pocket is highlighted, and the structure of PK9323 is shown. (B) Crystal structure of the CBP bromodomain compound 17 complex (PDB ID: 4NR5). The cross-section of the occupied binding pocket is highlighted, and the structures of compounds 17 and 33 are also shown. (C) Chemical structure of compounds 1–4. (D) Representative Western blot (WB) results of PK9323 and compounds 1–4 on inducing p21 protein expression from three independent experiments. NCI-H1299 (p53Y220C) stable cells were treated with the indicated compounds at the indicated concentrations for 24 h. (E) Chemical structure of compounds 5–9. (F) Representative WB results of compounds 5–9 on inducing p21 protein expression from three independent experiments. NCI-H1299 (p53Y220C) stable cells were treated with the indicated compounds at the indicated concentrations for 24 h. (G) Quantification of p21 induction (from panels D and F) by PK9323 and compounds 1–9 at the 24 h time point, following 5 μM treatment (from three independent experiments). (H) Left: representative WB results of the p53K382ac level following the treatment with the indicated compounds at 5 μM for 8 h from three independent experiments. NCI-H1299 (p53Y220C) stable cells were treated with the indicated compounds for 8 h, followed by endogenous immunoprecipitation (IP) FLAG pulldown. Right: quantification of the normalized p53K382ac level (p53K382ac level over total p53 protein level) following treatment with the indicated compounds at 5 μM for 8 h (from three independent experiments).

Figure 2.

Discovery and characterization of MS78, an effective p53Y220C AceTAC. (A) Chemical structure of MS78, derived from compound 7. (B) Comparison of the normalized p53K382ac level between compound 7 and MS78 in NCI-H1299 (p53Y220C) cells treated with the indicated compounds at 0, 1, 3, or 10 μM for 24 h. Results shown are the mean values ± SD from three independent experiments (** P < 0.01). (C) Left: WB results of the p53K382ac level in NCI-H1299 (p53Y220C) cells treated with MS78 at the indicated concentrations at 24 h. Results shown are representative of three independent experiments. Right: quantification of the normalized p53K382ac level on the left. Results shown are the mean values ± SD from three independent experiments. (D) WB results of the p53K382ac level in NCI-H1299 (p53Y220C) cells treated with 10 μM MS78 at the indicated time points. Results shown are representative of three independent experiments. (E) WB result of the p53K382ac level at the indicated time points post treatment of NCI-H1299 (p53Y220C) cells with MS78 at 10 μM for 6 h. Results shown are representative of three independent experiments. (F) Left: WB results of MS78-mediated p53-p300 interaction via p53-FLAG pull-down in NCI-H1299 (p53-null) and NCI-H1299 (p53Y220C) cells treated with MS78 at the indicated concentrations for 24 h. Results shown are representative of three independent experiments. Right: quantification of normalized p300 and p53K382ac levels from three independent experiments shown on the left. (G) Left: WB results of p53-FLAG pull-down after treatment of NCI-H1299 (p53Y220C) cells with MS78 at the indicated concentrations and with control siRNA or p300 siRNA. Results shown are representative of three independent experiments. Right: quantification of normalized p300 and p53K382ac levels from three independent experiments shown on the left. (H) Left: WB results of p53-FLAG pulldown after treatment of NCI-H1299 (p53Y220C) cells with MS78 alone at 1 μM, compound 33 alone at 10 μM, or compound 33 pretreatment at 10 μM for 2 h, followed by MS78 treatment at 1 μM for 18 h. Results shown are representative of four independent experiments. Right: quantification of the p300 and normalized p53K382ac levels from four independent experiments shown on the left (***P < 0.001).

Figure 3.

MS78 inhibits cell growth in NCI-H1299 p53Y220C cells but not in NCI-H1299 p53-null cells. Cell viability of MS78, PK9328, and compound 33 in (A) NCI-H1299 p53-null and (B) NCI-H1299 p53Y220C cells. The cells were treated with the indicated compounds at the indicated concentrations for 72 h. The mean values ± SD from three independent experiments are shown. (C) Left, representative WB results of the p53K282ac level induced by MS78, PK9328, and compound 33 at 0, 1, 3, and 10 μM in NCI-H1299 p53-null and NCI-H1299 p53Y220C cells (24 treatment). Right, quantification of the p53K382ac protein level normalized to Vinculin from two independent experiments. Cell viability of MS78, PK9328, and compound 33 in NCI-H1299 p53Y220C cells treated with (D) control siRNA or (E) p300-siRNA. The cells were treated with siRNA for 72 h and then treated with the indicated compounds at the indicated concentrations for 72 h. The mean values ± SD from three independent experiments are shown. (F) Left, representative WB results of the p53K282ac level in NCI-H1299 p53Y220C cells treated with control siRNA or p300-siRNA and then treated with MS78, PK9328, or compound 33 at 0, 1, 3, and 10 μM for 24 h. Right, quantification of the p53K382ac protein level normalized to Vinculin from two independent experiments.

Figure 4.

MS78 effectively inhibits cell growth in cancer cell lines that endogenously express p53Y220C and is nontoxic in WT p53 cells. (A) Cell viability results of MS78, PK9328, and compound 33 in BxPC3 cells, which were treated with DMSO or the indicated compounds at the indicated concentrations for 72 h. The mean value ± SD for each concentration point (in technical triplicates from three biological experiments) is shown in the curves. GraphPad Prism 8 was used in analysis of raw data. (B) Clonogenic assay results of MS78, PK9328, and compound 33 in BxPC3 cells treated with DMSO or 1, 3, or 10 μM of the indicated compounds for 14 days. Cells were fixed and stained with crystal violet, and the images are representative of two independent experiments. (C) Left: representative WB results of the p53K382ac protein level in BxPC3 cells treated with MS78, PK9328, or compound 33 at 0, 1, 3, or 10 μM for 24 h. Right: quantification of the normalized p53K382ac level in BxPC3 cells from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Cell viability results of MS78, PK9328, and compound 33 in NUGC-3 cells treated with DMSO or the indicated compounds at the indicated concentrations for 72 h. The mean value ± SD for each concentration point (in technical triplicates from three biological experiments) is shown in the curves. GraphPad Prism 8 was used in analysis of raw data. (E) Clonogenic assay results of MS78, PK9328, and compound 33 in NUGC-3 cells treated with DMSO or 1, 3, or 10 μM of the indicated compounds for 14 days. Cells were fixed and stained with crystal violet, and the images are representative of two independent experiments. (F) Left: representative WB results of the p53K382ac protein level in NUGC-3 cells treated with MS78, PK9328, or compound 33 at 0, 1, 3, or 10 μM for 24 h. Right: quantification of the normalized p53K382ac level in NUGC-3 cells from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (G) Cell viability assay results of MS78, PK9328, and compound 33 in U-2OS cells treated with the indicated compounds at the indicated concentrations for 72 h. The mean value ± SD for each concentration point (in technical triplicates from two biological experiments) is shown in the bar graph. (H) WB results of the p53K382ac protein level in U-2OS cells treated with MS78, PK9328, or compound 33 at 0, 1, 3, and 10 μM for 24 h. WB results shown are representative of two independent experiments.

Figure 5.

The p53Y220C AceTAC MS78 induces downstream signaling of canonical and noncanonical p53 pathways. (A) Heatmap enrichment in high-confidence p53-target genes. NCI-H1299 p53-null and p53Y220C cell lines were treated with DMSO or 10 μM MS78 for 24 h in quadruplicate. (B) Volcano plot of differential gene expression (DGE) of upregulated protein and pathways upon 10 μM MS78 treatment compared to DMSO for 24 h in NCI-H1299 p53Y220C cells. (C) Volcano plot of DGE of upregulated protein and pathways upon 10 μM MS78 treatment in NCI-H1299 p53Y220C cells compared to 10 μM MS78 treatment in NCI-H1299 p53-null cells and DMSO treatment in both cell lines. (D) Enrichment plot of significant genes upregulated from the 343 high-confidence p53-target genes (q-value < 0.01, normalized enrichment score (NES) = 1.52) in NCI-H1299 p53Y220C cells treated with 10 μM MS78 versus DMSO for 24 h in quadruplicate. (E) KEGG pathway analysis of upregulated protein and pathways upon 10 μM MS78 treatment compared to DMSO for 24 h in NCI-H1299 p53Y220C cells. (F) KEGG pathway analysis of upregulated protein and pathways upon 10 μM MS78 treatment in NCI-H1299 p53Y220C stable cell line compared to DMSO treatment in NCI-H1299 p53Y220C cells, DMSO treatment in NCI-H1299 p53-null cells, and 10 μM MS78 treatment in NCI-H1299 p53-null cells. The values of log₂ fold change and log₁₀ p-values in panels B and C were generated from four independent experiments.