The mechanisms by which cells respond and adapt to oxidative stress are largely unknown, but are key to developing a rationale for cancer therapies that target antioxidant pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation in myeloid dysplastic syndrome and acute myeloid leukemia. Whilst the mechanism of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent modification of cysteine residues in the core domain of mutant-p53 protein, we report that the anti-neoplastic capacity of APR-246 lies predominantly in depletion of low-molecular weight thiols such as free glutathione and cysteine.
Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of glutathione and mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was increased through loss of key enzymes in mitochondrial one-carbon metabolism, SHMT2 and MTHFD1L, while overexpression of components of Complex I of the electron transport chain and mitochondrial ribosome resulted in diminished efficacy. Whole-cell and mitochondrial proteomics analyses indicated an upregulation of proteins involved in iron-sulfur cluster biogenesis (eg. FDX1) and Complex I. Glutathione, acetyl-CoA and NADH levels were also depleted in APR-246 treated cells.
Importantly, we found that APR-246 targets low-molecular weight thiols in the mitochondria of cancer cells, which leads to a reduction in iron-sulfur cluster biogenesis. We propose that this in turn mediates a mitochondrial stress response, activating ATF4 and increasing mitochondrial biogenesis in an attempt to protect against oxidative stress. This work not only details novel determinants of APR-246 activity in cancer cells, but also provides a clinical roadmap for targeting antioxidant pathways in tumors – beyond targeting mutant-p53 tumors.