Abstract 5523: Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts

Abstract

Abstract p53 is the most frequently mutated gene in human cancer, and the p53R175 mutant is the third most frequently found missense mutant that confers resistance to anticancer drugs and apoptosis. Therefore, small-molecule reactivation of mutant p53 function(s) represents an important and novel anticancer strategy. We have previously reported that induction of oxidative protein modifications primarily underlies the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). Here, we describe the identification of Baylis-Hillman (BH) adducts as potent and novel mutant p53 reactivators through cell-based, high-throughput small-molecule screening procedures. An extensive structure-activity relationship studies indicated that carbon-carbon double bond and the presence of an electron-withdrawing substituent at the aromatic ring were essential for the activity. Cell lines used in this study includes SKBR3, AU565 (p53R175H), MCF7 (p53 wt), MDA-MB-231 (p53del), MDA-MB-468 (p53R273H) and MCF10a normal breast epithelial cells. BH adducts displayed preferential cytotoxicity against cancer cells expressing the allele-specific R175H mutation (SKBR3 and AU565) in comparison with the wt p53 or p53-null cancer cell lines. Additionally, the BH adducts did not show significant cytotoxicity against normal cells (MCF10a). Further, we developed isogenic cell lines expressing the p53R175H in the p53-null H1299 lung cancer cells and evaluated their sensitivities towards BH adducts; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of BH adduct stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that BH adduct treatment of p53R175H cells resulted in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed with BH adduct in a time and concentration-dependent manner. Other data showed that BH adduct restores the wt-structure and function by covalently binding with the mutant p53R175H. In conclusion, BH adduct is an attractive lead compound for further development because of its potent p53 reactivation of nonfunctional mutant protein (supported by CPRIT grants RP130266 and RP170207 to KSS). Citation Format: Surendra R. Punganuru, Viswanath Arutla, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5523.