Abstract 4053: Dynamic combinatorial p53 post-translational modifications regulate temporal transcription program and cell fates

Abstract The tumor suppressor p53 is activated by a range of cellular stresses including DNA damage, oncogenic signals, and nutrient deprivation. Upon activation, p53 transcriptionally upregulates an extensive repertoire of genes including those affecting cell fates such as cell cycle arrest, senescence, and apoptosis. What remains unclear is how information is conveyed through p53 to elicit the appropriate downstream responses according to the nature of the upstream stress. Changes in p53 temporal expression dynamics as well as specific p53 post-translational modifications (PTMs) are known to differentially regulate target gene induction. However, with over 100 known PTMs possible on p53, technical limitations have restricted the ability to detect the combinations of modifications on whole p53 molecules. Using a state-of-the-art mass spectrometry method (I2MS) which enabled the first measurement of individual intact human p53 proteins, we have begun to uncover the landscapes of combinatorial PTMs from different p53 activation regimes in cancer cell lines. Together with complementary p53 modification specific antibodies we identified changes in PTM patterns which were both stimuli and time dependent. For example, we found that in response to double-strand DNA breaks, p53 exhibits pulses of expression. Furthermore, p53’s post-translational modification state is altered between successive pulses, which we ascertained the most prominent temporal change in p53 PTMs in response to DNA damage to be a reduction of multiple p53 lysine acetylations. Inhibition of the deacetylase HDAC1, but not SIRT1, prevented the reduction in p53 acetylation, causing overexpression of senescence and apoptotic genes, and corresponding cell fates. Finally, we abrogated lysine acetylation by generating KR (lysine to arginine) mutations at single or multiple sites on p53, and found that acetylation sites are functionally non-redundant, with specific combinations exerting unique transcriptional effects on p53 target gene inductions. These results demonstrate an interplay between dynamics and modifications as a mechanistic strategy for cellular hubs like p53 to temporally organize multiple processes in individual cells. In addition, it highlights the importance of accounting for dynamical combinations of p53 PTMs in order to achieve the desired cellular response and efficacy of p53-mediated cancer therapies. Citation Format: Dan Lu. Dynamic combinatorial p53 post-translational modifications regulate temporal transcription program and cell fates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4053.