TP53 Knockout Cell Line-293T

Ewing sarcoma is a pediatric cancer driven by the EWS-ETS transcription factor fusion oncoprotein, which has a stable genomic background. Most tumors express wild-type TP53, so therapies targeting the p53 pathway will benefit most patients. To discover specific targets against TP53 wild-type Ewing sarcoma, researchers used a genome-scale CRISPR-Cas9 screen and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies. ATSP-7041, a stapled peptide inhibitor of MDM2 and MDM4, showed antitumor efficacy in vitro and in multiple mouse models. P5091, a USP7 inhibitor, and GSK2830371, a Wip1/PPM1D inhibitor, reduced the viability of Ewing sarcoma cells. Combinations of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic effects on the p53 pathway. Simultaneous knockout of TP53 rescued the effects of inhibitors including the specific USP7 inhibitor XL-188, highlighting the importance of intact p53 for the observed cytotoxic activity.

Three TP53 wild-type cell lines were infected with CRISPR-Cas9 constructs targeting TP53, and loss of TP53 was demonstrated by a reduction in the increase in p53 protein levels after etoposide treatment (Figure 1 A). Treatment of TP53 knockout cells showed that loss of TP53 completely rescued the cytotoxic effects of ATSP-7041, indicating that the drug has on-target activity (Figure 1 B) and that the response to MDM2/MDM4 inhibition is dependent on intact p53. Similarly, the Wip1 inhibitor GSK2830371 was less effective in TP53 knockout cells than in control cells, suggesting that GSK2830371 has on-target activity (Figure 1 C). However, TP53 knockout did not protect cells from the effects of P5091, suggesting a p53-independent mechanism or that this molecule may have off-target effects (Figure 1 D). Simultaneous loss of TP53 effectively rescued the cytotoxic effects of USP7 knockout, a phenomenon also observed with PPM1D knockout ( Figure 1 , E–G). XL-188 primarily reduced viability in TP53 wild-type Ewing sarcoma cell lines, with particularly strong effects observed in TC32 cells ( Figure 1 H). Surprisingly, TP53 knockout completely reversed the cytotoxic effects of XL-188 ( Figure 1 I), supporting the requirement for functional p53 to be required for the USP7 inhibition response observed in Ewing sarcoma.

Figure 1. Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (Stolte, Björn, et al. 2018)