Restoration of the Tumor Suppressor Function of Y220C-Mutant p53 in vitro by Rezatapopt (PC14586), a Small Molecule Reactivator [in vitro]

Restoration of the tumor suppressor function of tumor-associated p53 mutants, including tumors harboring the TP53 Y220C mutation, has posed a significant challenge and remains an attractive target for therapeutic discovery. Here, we describe a clinical candidate Rezatapopt (PC14586) designed to stabilize the p53 Y220C mutant protein and restore wild-type (WT) p53 function. PC14586 induced global gene expression changes in the cell lines harboring the TP53 Y220C mutation (NUGC-3 and T3M-4) but not in cells lacking p53 Y220C-mutant protein (NUGC-3 KO and SJSA-1). Extensive transcriptomics analysis of cells harboring the TP53 Y220C mutation following PC14586 treatment revealed the induction of p53 direct target genes, including protein-coding genes and long non-coding RNAs (lncRNAs), and repression of cell cycle progression-associated genes in a distinct temporal pattern. Our data demonstrate that PC14586 administration inhibited cell proliferation in vitro via the induction of WT p53 transcriptional signatures, including a sustained transcriptional repression of genes involved in cell cycle regulation. Following these investigations and additional in vivo studies, rezatapopt (PC14586) was identified as a clinical candidate and is currently being evaluated in the registrational Phase 2 PYNNACLE study. Overall design: To investigate the selectivity and robustness of PC14586 in reactivating the p53 Y220C mutant protein to p53 WT function in cells, we treated 4 different cells lines - NUGC-3 (TP53 Y220C), T3M-4 (TP53 Y220C), NUGC-3 KO (TP53 KO), and SJSA-1 (TP53 WT) – with PC14586 (5 µM) versus DMSO at two time points (5h and 16h) in 3 biological repeats for each. We performed comparative gene expression profiling analysis of RNA-seq data from each of 4 cell lines with PC14586 versus DMSO treatments (n=3 each).