The determinants of sensitivity and mechanism of action of eprenetapopt (APR-246)

Abstract

The tumour suppressor gene TP53 is mutated in over half of all cancers, resulting in mutant p53 protein accumulation and poor patient survival. Decades after the discovery that p53 plays a central role in the human body’s defence against cancer, the development of effective therapeutic strategies to target mutant p53 cancers still remains an unmet need in oncology. Eprenetapopt (also known as APR-246 and PRIMA-1MET) is currently in clinical development as a mutant p53 reactivator. The reported mechanism of action of eprenetapopt is to restore wild-type p53 functionality to mutant p53 in cancer cells, triggering p53-dependent cell cycle arrest and caspase- dependent apoptosis. As a result, clinical investigation of eprenetapopt has focused on TP53-mutated cancers, relying solely on TP53 mutation status for patient selection into clinical trials. However, the mechanism of action of eprenetapopt remains contentious, as evidence demonstrates that eprenetapopt maintains strong potency in cancer cells with no mutant p53. Herein, utilising novel unbiased approaches, including CRISPR perturbation screening, metabolomics, proteomics and pan-cancer cellular-feature profiling, this thesis demonstrates two major innovations regarding the determinants of sensitivity and mechanisms of action of eprenetapopt. First, the expression of SLC7A11, which encodes the functional subunit of the cystine- glutamate antiporter, not TP53-mutation status, is the major determinant of sensitivity to eprenetapopt. Second, eprenetapopt triggers a caspase-independent, iron- dependent form of cell death known as ferroptosis, following glutathione depletion and inhibition of iron-sulfur cluster biogenesis. Together, this thesis provides a roadmap for broadening the clinical utility of eprenetapopt as a ferroptosis inducer in haematological and solid malignancies, beyond TP53-mutant cancers.