Abstract 5288: Development of lead mutant-p53 reactivating peptides towards clinical application

Abstract

Abstract p53 is the most extensively studied tumor suppressor. It serves as a major barrier against malignant transformation and the p53 pathway is inactivated in almost all human tumors. A large percentage of tumors inactivate p53 by point mutations in the DNA binding domain (DBD) of the protein. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, improperly folded form. Previously, we reported the use of phage display technology to select for peptides that reactivate mutant p53 by stabilizing a wt protein conformation, taking advantage of a unique combination of selection steps (Tal P, at al Oncotarget. 2017 Jan 3;8(1):164-178). We now describe further progress in the development of one lead peptide, pCAP-250, which contains 9 amino acids and a myristic fatty acid at its N-terminus. The pCAP-250 sequence bears 100% homology to RAD9, a known p53 interacting protein. We show physical interaction between pCAP-250 and the p53DBD. Characterization of this interaction by NMR reveals a significant chemical shift of amino acids located in the p53DBD L1 loop and Helix-2, two regions critical for p53 transcriptional activity and interaction with DNA. We also report the optimization of our lead sequence by rational design to yield more potent derivatives with higher efficacy in cancer cell elimination and longer in vivo half-life. RNA-seq analysis shows that our lead peptides induce robust activation of p53 target genes. Furthermore, we report that in addition to p53 activation, pCAP-250 also has a p53-independent anti-cancer activity. Overall, our lead peptides cause specific cell death in a wide spectrum of mutant p53 expressing cell lines, with an IC50 of 0.1-12µM, while having no effect on non-transformed cells at these concentrations. The pharmacokinetic profile of pCAP-250 and its derivatives shows that they are completely stable in human serum and have plasma half-life of 1.5-10h in vivo when administered IV or subcutaneously, presumably due to interaction with albumin through the fatty acid moiety. Remarkably, our lead peptides elicit remarkable regression of very aggressive tumors in several preclinical mouse models of renal, colon, pancreatic and ovarian cancer. In vivo efficacy can be achieved through several routes of administration, including intra-tumor injection as well as systemic subcutaneous continuous slow release and subcutaneous bi-daily injections. Thus, such peptides might serve as novel therapeutic agents for p53-mutated human cancer. Citation Format: Perry Tal, Moshe Oren, Orna Palgi, Varda Rotter, KWANGIL JEONG. Development of lead mutant-p53 reactivating peptides towards clinical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5288.