Mechanistic Insights into DNA Recognition by FOXO4 and Development of a Peptide Targeting Senescent Cells via FOXO4-p53 Inhibition

Abstract

This thesis investigates the molecular mechanisms underlying the function of the human forkhead box O 4 (FOXO4) transcription factor, elucidates its DNA recognition mechanism, and develops a novel therapeutic approach for targeting senescent cells, highlighting its potential impact on aging biology. First, we explore the mechanism by which FOXO4 differentially recognizes target and non-target DNA sequences despite exhibiting similar binding affinities. This selectivity is crucial for precise transcriptional regulation but has remained unclear. Through comprehensive NMR-based analyses, particularly paramagnetic relaxation enhancement (PRE) experiments, we demonstrate that the conserved region 3 (CR3) transactivation domain (TAD) plays a crucial regulatory role in DNA sequence discrimination. Our findings reveal that CR3 remains proximal to the forkhead domain (FHD) when non-target DNA is present, effectively occluding the non-target DNA-binding interface. In contrast, CR3 is released upon FHD binding to target DNA, as the FHD forms a stable complex with the DNA. Furthermore, 15N relaxation measurements indicate that FHD exhibits flexibility when bound to non-target DNA but adopts a more rigid conformation upon target DNA binding. These interactions are primarily mediated by electrostatic forces, as evidenced by their sensitivity to salt concentrations. This sensitivity provides insight into how FOXO4 can navigate the cellular environment to locate and bind its target sequences amidst the vast genomic landscape. The dynamic interplay between CR3 and FHD represents a novel mechanism for transcription factor target selection that extends conventional DNA-protein recognition models. Next, we apply our understanding of FOXO4 interactions to address the challenge of cellular senescence in aging. Senescent cells, characterized by permanent cell cycle arrest and secretion of inflammatory factors, contribute significantly to age-related pathologies and cancer recurrence following chemotherapy. We aim to target the FOXO4-p53 interaction, essential for senescent cell survival and potentially druggable. Through systematic biophysical methods, including NMR spectroscopy and cellular experiments, we determined critical regions within the p53 TAD involved in FOXO4 binding. This detailed structural knowledge enabled us to design CPP-CAND, an optimized cell-penetrating peptide inhibitor targeting the FOXO4-p53 interaction. Our peptide demonstrated remarkable selectivity in disrupting FOXO4-p53 nuclear foci and inducing apoptosis in senescent cells while sparing non-senescent counterparts. Notably, CPP-CAND proved effective against senescent cancer cells induced by common chemotherapy treatments with doxorubicin and cisplatin, suggesting potential applications as an adjuvant therapy to prevent cancer recurrence. Mechanistic studies confirmed that CPP-CAND functions by activating caspase-mediated apoptosis pathways specifically in senescent cells, offering advantages in selectivity, efficacy, and cost-effectiveness over existing senolytic approaches. Collectively, this research advances our understanding of transcription factor biology and demonstrates how mechanistic insights at the molecular level can be translated into targeted therapeutic strategies. We have elucidated a novel regulatory mechanism for FOXO4's DNA target selection and leveraged our understanding of protein-protein interactions to develop a promising senolytic agent. The designed peptide inhibitor offers significant potential for addressing challenges in aging biology and cancer therapy through specific disruption of the FOXO4-p53 interaction, thereby opening new avenues for intervention in age-related diseases and chemotherapy-induced senescence.