The SLC7A11/Glutathione Axis is a Therapeutic Target in Mutant P53 Cancers.

Introduction: Effective therapeutic strategies to target mutant-p53 cancer cells remain an unmet medical need. Several compounds, including APR-246, have been developed that restore wild-type activity to mutant-p53 protein. APR-246 has also been shown to target anti-oxidant pathways in a p53-independent manner. We investigated whether this aspect of APR-246 activity could be exploited further to specifically target mutant-p53 cancers.

Materials and Methods: Effects of mutant-p53 and APR-246 treatment on cell growth, viability, expression and activity of anti-oxidant response genes and transcription factor binding (ChIP-PCR), anti-oxidant levels and oxidative stress markers were investigated in oesophageal cancer cell lines and isogenic p53-null/mutant-p53 cDNA expression and CRISPR knockout models. Both genetic and pharmacological approaches were used to interrogate the effects of inhibiting SLC7A11 in the presence and absence of mutant-p53 in vitro and in cell line and patient derived xenograft models.

Results and Discussion: We found that mutant-p53 impairs the function of the transcription factor, NRF2, to regulate genes involved in cellular detoxification in response to oxidative stress. We show that mutant-p53 accumulation across multiple tumour types represses the transcription of NRF2 target genes, particularly SLC7A11, a key component of the glutamate/cysteine exchanger, system xCT-. Downgregulation of SLC7A11 by accumulation of mutant-p53 results in reduced cystine import, lowering endogenous stores of the major cellular anti-oxidant, glutathione. This in turn raises basal reactive oxygen species levels and predisposes cells to oxidative damage. Notably, genetic knockdown or pharmacological inhibition (erastin and sulfasalazine) of SLC7A11 creates a synthetic lethal interaction with mutant-p53 accumulation in both in vitro and in vivo models, a therapeutic paradigm similar to the use of PARP inhibitors in BRCA deficient cancers. Moreover, we found that APR-246 depletes cellular glutathione and induces significantly higher amounts of ROS in mutant-p53 cancer cells compared with normal cells. This leads to lipid peroxidation of mitochondrial membranes and the release of matrix contents, culminating in apoptotic cell death. APR-246-induced cytotoxicity could be rescued by cysteine or glutathione replacement, or with lipophilic antioxidants. APR-246 also reverses the suppressive effect of mutant-p53 on NRF2, leading to up-regulation of SLC7A11, a potential negative feedback mechanism that antagonises drug activity. In extension, we demonstrate that antagonising SLC7A11 activity in combination with APR-246 selectively and synergistically inhibits mutant-p53 cancer cells.

Conclusion: We propose that accumulation of mutant-p53 protein in cancer cells, through its repressive effects on NRF2 function creates an ‘Achilles heel’ that can be targeted by further perturbations of the SLC7A11/glutathione axis.