p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

Abstract The transcription factor and tumor suppressor p53 is one of the most frequently mutated genes in cancer and has been difficult to target therapeutically due to its intrinsically disordered regions. The hotspot mutation p53Y220C, a mutation thermodynamically destabilizing p53, creates a unique extended crevice on the surface of the protein for which chemical matter has been identified over the last years. Advanced p53Y220C stabilizers reconstitute p53Y220C to its wildtype conformation, thereby restoring p53’s role in target gene expression and inhibiting the growth of p53Y220C mutant cancer cell lines. We hypothesized that direct recruitment of the transcriptional elongation machinery to p53Y220C and its target genes may potentiate effects beyond p53 protein stabilization alone. We leveraged induced proximity to discover bifunctional molecules, p53Y220C- t argeted PR oximity I nduced M odulators of E xpression (tPRIMEs), that specifically recognize the BET bromodomain proteins and induce stable ternary complexes with p53Y220C. p53Y220C-tPRIMEs potently inhibit proliferation and induce apoptosis of p53Y220C mutant cancer cell lines to a greater extent than the parental ligands alone or in combination. Gene expression analyses revealed that p53Y220C-tPRIMEs induce an increase in p53 target gene expression compared to parental binders. The superior antiproliferative activity, enhanced apoptosis, and increased p53 target gene expression are dependent on ternary complex formation. These data strongly suggest that a p53Y220C-tPRIME-mediated induced proximity approach between transcriptional regulators and p53Y220C - in contrast to p53 stabilization alone - can modulate the cell fate control from cell cycle inhibition to an apoptotic response, providing a compelling therapeutic modality for p53 mutant cancers.