Journal: Cell Reports Medicine
Article Title: A triple-action PROTAC for wild-type p53 cancer therapy
doi: 10.1016/j.xcrm.2025.102467
Figure Lengend Snippet: Potent and selective cytotoxicity of TAPTAC1 correlates with degradation of BET proteins and reactivation of the p53 pathway (A) To assess the relative potency and selectivity of TAPTAC1, we compared its anti-cancer activity in culture with A1874, a 2-in-1 PROTAC comprising the selective HDM2 inhibitor RG7388 and JQ1, and the TAPTAC1 F19A point mutant, which abrogates interaction of the stapled p53 peptide component of the chimera with HDM2 and HDMX. (B–D) In SJSA-1 cells that have genetic amplification of HDM2 but little to no HDMX expression, TAPTAC1 exhibits marginally increased potency compared to A1874, as assessed by viability assay (B). In SJSA-X cells, engineered to express HDMX, the dual-targeting capability of TAPTAC1 results in markedly enhanced potency compared to A1874 (C). In Saos-2 cells, which lack p53, no p53/HDM2/HDMX-based activity or selectivity is evident, consistent with the compounds functioning similarly based on residual BET inhibitor activity alone (D). In each cell line, F19A point mutagenesis likewise abrogates the p53-dependent activity of TAPTAC1, further highlighting its specificity of action. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (E) Upon treatment of SJSA-X cells with 100 nM TAPTAC1, we observed prompt degradation of BRD4 within 8 h, coinciding with time-dependent upregulation of p53, which peaked at 12 h and triggered both a surge in p21 and counter-elevation of HDM2 and HDMX by 24 h. The reduction of p53 levels observed between 12 and 24 h is consistent with the characteristic negative feedback loop of the p53 pathway. Time-dependent western blot analyses were performed twice using independent SJSA-X cell cultures and compound treatment. NT, no treatment. (F) Quantitative proteomics revealed that TAPTAC1 treatment (1 μM, 24 h) of SJSA-X cells caused a striking reduction of BET protein levels (cyan, e.g., BRD2-4) and marked upregulation of p53 pathway proteins (red, e.g., p53 [TP53], p21 [CDKN1A], HDM2 [MDM2], and HDMX [MDM4]). Quantitative proteomic analysis was performed using three biological replicates representing independent cultures and treatment. ∗, isoform. See also , , and .
Article Snippet: The human BRD4 BD1 domain (residues 48–168) in the pNIC28Bsa4 vector (Addgene) was expressed in BL21(DE3) E. coli in LB medium containing 50 mg/mL kanamycin.
Techniques: Activity Assay, Mutagenesis, Amplification, Expressing, Viability Assay, Western Blot, Quantitative Proteomics
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![Potent and selective cytotoxicity of TAPTAC1 correlates with degradation of BET proteins and reactivation of the p53 pathway (A) To assess the relative potency and selectivity of TAPTAC1, we compared its anti-cancer activity in culture with A1874, a 2-in-1 PROTAC comprising the selective HDM2 inhibitor RG7388 and JQ1, and the TAPTAC1 F19A point mutant, which abrogates interaction of the stapled p53 peptide component of the chimera with HDM2 and HDMX. (B–D) In SJSA-1 cells that have genetic amplification of HDM2 but little to no HDMX expression, TAPTAC1 exhibits marginally increased potency compared to A1874, as assessed by viability assay (B). In SJSA-X cells, engineered to express HDMX, the dual-targeting capability of TAPTAC1 results in markedly enhanced potency compared to A1874 (C). In Saos-2 cells, which lack p53, no p53/HDM2/HDMX-based activity or selectivity is evident, consistent with the compounds functioning similarly based on residual BET inhibitor activity alone (D). In each cell line, F19A point mutagenesis likewise abrogates the p53-dependent activity of TAPTAC1, further highlighting its specificity of action. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (E) Upon treatment of SJSA-X cells with 100 nM TAPTAC1, we observed prompt degradation of BRD4 within 8 h, coinciding with time-dependent upregulation of p53, which peaked at 12 h and triggered both a surge in p21 and counter-elevation of HDM2 and HDMX by 24 h. The reduction of p53 levels observed between 12 and 24 h is consistent with the characteristic negative feedback loop of the p53 pathway. Time-dependent western blot analyses were performed twice using independent SJSA-X cell cultures and compound treatment. NT, no treatment. (F) Quantitative proteomics revealed that TAPTAC1 treatment (1 μM, 24 h) of SJSA-X cells caused a striking reduction of BET protein levels (cyan, e.g., BRD2-4) and marked upregulation of p53 pathway proteins (red, e.g., p53 [TP53], p21 [CDKN1A], HDM2 [MDM2], and HDMX [MDM4]). Quantitative proteomic analysis was performed using three biological replicates representing independent cultures and treatment. ∗, isoform. See also , , and .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5843/pmc12765843/pmc12765843__gr2.jpg)
