Making sense from nonsense and missense : pharmacological rescue of mutant tumor suppressor p53

A large fraction of human tumors carry inactivating mutations in the TP53 tumor suppressor gene. Approximately 75% of these mutations are missense mutations and around 10% are nonsense mutations. TP53 inactivation allows evasion of cell death and rapid tumor progression. Restoration of wild type p53 protein expression in tumor cells can suppress tumor growth in vivo. Missense mutant p53 is often expressed at high levels in tumors. Therefore, mutant p53 is a promising target for novel targeted cancer therapy. APR-246 (PRIMA-1Met) is a low molecular compound previously discovered in our group. We have shown that APR-246 is converted to methylene quinuclidinone (MQ), a potent electrophile and Michael acceptor, binding covalently to cysteines in p53. MQ promotes refolding of mutant p53 to wild type conformation and restores normal function to mutant p53, resulting in induction of apoptosis and inhibition of xenograft tumor growth in mice. However, the underlying mechanism is not fully elucidated. APR-246 is currently tested in a phase II clinical trial in high-grade serous ovarian cancer. In paper I, we show that APR-246, via MQ, targets the Sec residue in the redox enzyme TrxR1 and inhibits the reducing activity of both recombinant TrxR1 in vitro and cellular TrxR1 in cells independently of p53 status. The inhibited TrxR1 maintains its ability to oxidase NADPH, which contributes to oxidative stress and cell death induced by APR-246. In paper II, we have confirmed the ability of aminoglycosides to induce translational readthrough of nonsense mutant TP53 and also shown that combination treatment with aminoglycosides and p53-Mdm2 inhibitors such as nutlin can enhance levels of full-length p53 and p53 activity in tumor cells carrying TP53 nonsense mutation. In paper III, we through data mining have identified 28 compounds with potential ability to target nonsense mutant TP53-carrying cancer cells. Among these, the known anticancer drug 5-FU and 5 other compounds were shown to induce full-length p53 protein in nonsense mutant TP53-carrying cancer cells. Full-length p53 protein induced by 5-FU is transcriptionally active as assessed by upregulation of several p53 target genes. In paper IV, we identified 65 compounds, from high-throughput screening and FACS-based screening of chemical libraries as candidate readthrough-inducing compounds (CRICs). Among these, 7 compounds were shown by Western blotting to induce full-length p53 protein in nonsense mutant TP53-carrying tumor cells. In conclusion, our study reveals that the missense mutant p53-reactivating compound APR246 inhibits TrxR1 and converts the enzyme to an NADPH oxidase. We have also shown that combination treatment with p53-Mdm2 inhibitors enhances the ability of aminoglycosides to restore expression and function of full-length p53 in cancer cells with nonsense mutant TP53. Furthermore, we identified 5-FU and several novel compounds as compounds that can rescue expression of full-length p53 in tumor cells with nonsense mutant TP53. Our studies shed further light on the mechanism underlying APR-246 induced cancer cell death and raise possibilities for nonsense mutant TP53-targeted cancer therapy in the future. Our results may ultimately facilitate the development of novel treatment for tumors carrying missense or nonsense mutant TP53. LIST OF SCIENTIFIC PAPERS I. Peng, X. *, Zhang, M.Q. *, Conserva, F. *, Hosny, G. , Selivanova, G. , Bykov, V.J., Arner, E.S. , and Wiman, K.G. APR-246/PRIMA-1MET inhibits thioredoxin reductase 1 and converts the enzyme to a dedicated NADPH oxidase. Cell Death Dis (2013) 4, e881. (*these authors contributed equally). II. Zhang, M. *, Heldin, A. *, Palomar-Siles, M. , Ohlin, S. , Bykov, V.J.N. , and Wiman, K.G. Synergistic rescue of nonsense mutant tumor suppressor p53 by combination treatment with aminoglycosides and Mdm2 inhibitors. Front Oncol (2017)7, 323. (*these authors contributed equally). III. Zhang, M. , Palomar-Siles, M. , Heldin, A. , Bykov, V.J.N. , and Wiman, K.G. Rescue of nonsense mutant p53 in human tumor cells by compounds identified through data mining (Manuscript). IV. Zhang, M. , Heldin, A. , Bykov, V.J.N. , and Wiman, K.G. High-throughput screening for identification of novel compounds that induce translational readthrough of nonsense mutant TP53 (Manuscript). CONTENT