Julianne S. Funk

The tumor suppressor p53 is the most frequently mutated protein in tumors and a target for drug development. More than 2000 cancer-associated p53 missense mutations have been reported, most of them located in the DNA-binding domain (DBD). Due to the low intrinsic thermostability of the latter, they often lead to unfolding at physiological temperature. Stabilizing the DBD with small molecules has been shown to be effective in reactivating the cavity-creating cancer mutant Y220C. Unfortunately, the majority of p53 mutants seem to lack druggable binding pockets for small molecules. Here we show that a designed ankyrin repeat protein (DARPin) that binds to the p53 DBD stabilizes temperature-sensitive (TS) p53 cancer mutants, thereby compensating for mutation-induced loss of stability. We determined high-resolution crystal structures of multiple DARPin–mutant p53 complexes, providing mechanistic insights into this mode of stabilization. Reporter gene assays across a comprehensive panel of cancer-associated mutants revealed reactivation of the majority of TS mutants, whereas DNA-contact mutants and those with local misfolding of the DNA-binding surface remained inactive, as expected. We demonstrate that this reactivation induces the transcription of canonical p53 target genes and elicits antiproliferative effects in cancer cell lines. A combination of this DARPin with an mRNA/lipid nanoparticle-based transfection approach may have the potential to reactivate most TS p53 mutants and resensitize cancer cells to chemotherapy.

The cavity-creating p53 cancer mutation Y220C, which accounts for an estimated 125,000 new cancer cases per year, serves as an excellent paradigm for the development of mutant p53 reactivators. Several molecules that reactivate this thermolabile cancer mutant by targeting the mutation-induced crevice have been developed, and one of them, rezatapopt, is currently in clinical trials. The less frequently occurring Y220N and Y220S mutations are even more destabilizing than Y220C but create a similar surface crevice, raising the question of whether cancer patients with these mutations might also benefit from rezatapopt treatment. Here, we show that rezatapopt also binds to the Y220N and Y220S mutants, with nanomolar affinity, resulting in a full recovery of wild-type-like stability for the latter. High-resolution crystal structures of all three mutants bound to rezatapopt revealed a conserved binding mode, highlighting key interactions, including multipolar interactions of a fluorine substituent at a chiral center with the protein backbone. Consistent with the biophysical and structural data, rezatapopt reactivated p53 signaling in both Y220C and Y220S mutant cells by restoring the folded conformation and transcriptional activity, leading to anti-proliferative effects and apoptosis, albeit requiring higher compound concentrations in Y220S cells. The Y220N mutant, despite exhibiting high-nanomolar affinity for rezatapopt and substantial stabilization, did not show noticeable effects in cells at the concentrations tested, as rezatapopt binding resulted in only partial compensation for the mutation-induced loss of stability, for which we provide a structural explanation. Our data suggest that the development of clinical pan-Y220C/N/S reactivators, which could benefit an additional 10,000 patients per year, is challenging but not impossible.

Infection of cells with high-risk strains of the human papillomavirus (HPV) causes cancer in various types of epithelial tissue. HPV infections are responsible for ~4.5% of all cancers worldwide. Tumorigenesis is based on the inactivation of key cellular control mechanisms by the viral proteins E6 and E7. The HPV E6 protein interacts with the cellular E3 ligase E6AP, and this complex binds to the p53 DNA-binding domain, which results in degradation of p53. Inhibition of this interaction has the potential to reactivate p53, thus preventing oncogenic transformation. Here we describe the characterization of a designed ankyrin repeat protein that binds to the same site as the HPV E6 protein, thereby displacing the E3 ligase and stabilizing p53. Interaction with the designed ankyrin repeat protein does not affect p53 DNA binding or the crucial MDM2 negative feedback loop but reactivates a p53-dependent transcriptional program in HeLa (HPV18-positive) and SiHa (HPV16-positive) cells, suggesting a potential therapeutic use.

The mutational landscape of TP53 , a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors duringDrosophilahematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiationin vivoand that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.