Abstract A046: Functional genetic screen of TP53 mutants reveals APR-246 sensitivity is mediated by p53 independent effects

TP53, often referred to as the guardian of the genome, is one of the most frequently mutated genes in human cancers. High grade serous ovarian cancer (HGSOC) has a TP53 mutation rate of up to 96%. Given its multi-faceted role in human cancers, there have been many efforts to therapeutically target mutant p53. APR-246 is a small molecule drug that is proposed to bind mutant p53 and promote its thermodynamic stabilization, allowing the tumor suppressor to regain wild-type-like conformation and functionality. However, the mechanism of this drug has remained a contentious topic, as it has been shown to act via various p53-independent mechanisms, including modulation of the redox system. To address this, we have performed a systematic functional genetic screening for the identification of APR-246 rescuable p53 mutants. Our approach leverages a pooled TP53 mutagenesis library containing ∼8,000 amino acid variants, spanning the entirety of the TP53 coding sequence. Cell based screenings against sub-lethal doses of APR-246 in two ovarian cancer cell lines, SKOV3 and OVCAR5, has revealed that sensitivity to this drug is cell-line dependent rather than mutant p53-dependent. Our results suggest that p53-independent factors such as intrinsic resistance to ferroptosis may play a more significant role in response to APR-246. We propose that resistance to ferroptosis provides cellular conditions which enable identification of p53 mutants that are sensitive to APR-246. This is evidenced by our results which show that genetic screen in OVCAR5, which is more intrinsically resistant to ferroptosis, uncovers a greater number of potentially sensitive mutants, including R175H, a mutant largely recognized as rescuable by APR-246. Further, we have identified GPX4, an anti-ferroptosis gene, as a key determinant of APR-246 sensitivity. Indeed, we demonstrate synergy between APR-246 and GPX4 inhibitor, ML-210. These studies support the hypothesis that APR-246 efficacy is largely attributable to p53-independent mechanisms and that attenuation of these off-target effect may reveal potentially sensitive p53 mutants. Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chien. Functional genetic screen of TP53 mutants reveals APR-246 sensitivity is mediated by p53 independent effects [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A046.