Impact of combined MRP1 inhibition and mutant p53-targeting compound APR-246.

Abstract

e14712 Background: The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. Mutant p53 protein is often expressed at high levels and accompanied with gain-of-function activities that promote tumor development and resistance towards conventional treatment. APR-246 is a mutant p53-reactivating small molecule undergoing a Phase III clinical study in myelodysplastic syndrome (MDS), and several phase II studies. APR-246 is non-enzymatically converted to its active product methylene quinuclidinone (MQ) which binds to cysteine residues in p53. This stimulates proper folding of p53's DNA-binding core domain, leading to cell death. APR-246 also exhibits pro-oxidant activity as the electrophile MQ binds and inactivates important antioxidants such as glutathione and thioredoxin reductase, which both are essential for cellular defense against oxidative and electrophilic stress. Methods: All results are in vitro experiments by LC-MS as well as experiments in cultured cells, including 14C-APR-246/MQ detection, cell viability measurements, LC-MS analysis, enzyme recycling measurements and Western blotting. Results: We have utilized 14C-labelled APR-246 to investigate the effect of APR-246 and its active moiety MQ in tumor cell cultures upon inhibition of efflux transporter multidrug resistance protein 1 (MRP1) or cystine-glutamate transporter (xCT). Transient downregulation or small molecule inhibitors (MK-571, reversan, sulfasalazin) targeting MRP1 or xCT increased drug content and shifted intra- and extracellular thiol status. Missense mutant TP53-carrying cells exhibited higher sensitivity to APR-246 and combination therapies. MRP1 inhibition showed strong synergy with APR-246 and increased intracellular levels of MQ conjugated to glutathione (GS-MQ). We found that GS-MQ conjugate formation is reversible, and suggest that the intrinsic reversibility of MQ adduct formation is an important aspect of the mechanism of action of APR-246. This reversibility may also in part account for the benign safety profile reported from clinical studies with APR-246. Conclusions: Sensitivity to APR-246 is affected by MRP1 efflux activity and the redox status, reflected in ratios of cysteine/cystine and reduced/oxidized glutathione (GSH/GSSG).