Rusin, S. | bioRxiv Science

bioRxiv2025

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

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 p53s 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-targeted PRoximity Induced Modulators of Expression (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.

cancer biology↗

bioRxiv2021

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent activity, suggesting that Ssn3 kinase activity negatively regulates Flo8. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that FLO8 and STP2, a transcription factor involved in amino acid utilization, are required for ssn3{Delta}/{Delta} phenotype, but that loss of the Ssn3 phosphosites identified in Flo8 was not sufficient to phenocopy the ssn3{Delta}/{Delta} mutant. These data highlight the spectrum of processes affected by the modulation of Ssn3 activity and underscore the importance of considering Ssn3 function in the control of transcription factor activities.

microbiology↗