The transcription factor p53 functions as a critical tumor suppressor by orchestrating a plethora of cellular responses such as DNA repair, cell cycle arrest, cellular senescence, cell death, cell differentiation, and metabolism. In unstressed cells, p53 levels are kept low due to its polyubiquitination by the E3 ubiquitin ligase MDM2. In response to various stress signals, including DNA damage and aberrant growth signals, the interaction between p53 and MDM2 is blocked and p53 becomes stabilized, allowing p53 to regulate a diverse set of cellular responses mainly through the transactivation of its target genes. The outcome of p53 activation is controlled by its dynamics, its interactions with other proteins, and post-translational modifications. Due to its involvement in several tumor-suppressing pathways, p53 function is frequently impaired in human cancers. In colorectal cancer (CRC), the TP53 gene is mutated in 43% of tumors, and the remaining tumors often have compromised p53 functioning because of alterations in the genes encoding proteins involved in p53 regulation, such as ATM (13%) or DNA-PKcs (11%). TP53 mutations in CRC are usually missense mutations that impair wild-type p53 function (loss-of-function) and that even might provide neo-morphic (gain-of-function) activities such as promoting cancer cell stemness, cell proliferation, invasion, and metastasis, thereby promoting cancer progression. Although the first compounds targeting p53 are in clinical trials, a better understanding of wild-type and mutant p53 functions will likely pave the way for novel CRC therapies.

Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response.

Mild and massive DNA damage are differentially integrated into the cellular signaling networks and, in consequence, provoke different cell fate decisions. After mild damage, the tumor suppressor p53 directs the cellular response to cell cycle arrest, DNA repair, and cell survival, whereas upon severe damage, p53 drives the cell death response. One posttranslational modification of p53, phosphorylation at Serine 46, selectively occurs after severe DNA damage and is envisioned as a marker of the cell death response. However, the molecular mechanism of action of the p53 Ser46 phospho-isomer, the molecular timing of this phosphorylation event, and its activating effects on apoptosis and ferroptosis still await exploration. In this essay, the current body of evidence on the molecular function of this deadly p53 mark, its evolutionary conservation, and the regulation of the key players of this response, the p53 Serine 46 kinases, are reviewed and dissected.

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Evasion of immune recognition by the innate and acquired immune system is a major principle of tumour cells and belongs to the hallmarks of cancer. Immune checkpoint inhibitor-based cancer therapies targeting the co-inhibitory receptors CTLA-4 or PD-1 have received enormous scientific and clinical attention during the last few years, because of promising clinical results observed in the treatment of different cancer entities including melanoma and cutaneous squamous cell carcinoma. However, the enthusiasm about the effects of the immune checkpoint inhibitors is muted as only a subfraction of patients shows a stable clinical response. To predefine the patient cohorts that may benefit from immune checkpoint therapy, rigorous biomarker analyses, which predict the response to these novel therapies, need to be performed. In addition, combination of immune checkpoint therapy with classical DNA-damaging chemotherapy or radiotherapy, which positively affects tumour neo-antigen presentation, appears to be a promising approach in optimizing patients' response. In this review, we briefly summarize important biomarkers for patient stratification and discuss the current limitations of these biomarkers in defining responders vs. non-responders to immune checkpoint therapy.

The DNA damage-responsive tumor suppressors p53 and HIPK2 are well established regulators of cell fate decision-making and regulate the cellular sensitivity to DNA-damaging drugs. Here, we identify Deleted in Azoospermia-associated protein 2 (DAZAP2), a small adaptor protein, as a novel regulator of HIPK2 and specifier of the DNA damage-induced p53 response. Knock-down or genetic deletion of DAZAP2 strongly potentiates cancer cell chemosensitivity both in cells and in vivo using a mouse tumour xenograft model. In unstressed cells, DAZAP2 stimulates HIPK2 polyubiquitination and degradation through interplay with the ubiquitin ligase SIAH1. Upon DNA damage, HIPK2 site-specifically phosphorylates DAZAP2, which terminates its HIPK2-degrading function and triggers its re-localization to the cell nucleus. Interestingly, nuclear DAZAP2 interacts with p53 and specifies target gene expression through modulating a defined subset of p53 target genes. Furthermore, our results suggest that DAZAP2 co-occupies p53 response elements to specify target gene expression. Collectively, our findings propose DAZAP2 as novel regulator of the DNA damage-induced p53 response that controls cancer cell chemosensitivity.

By forming specific functional entities, nuclear biomolecular condensates play an important function in guiding biological processes. PML biomolecular condensates, also known as PML nuclear bodies (NBs), are macro-molecular sub-nuclear organelles involved in central biological processes, including anti-viral response and cell fate control upon genotoxic stress. PML condensate formation is stimulated upon cellular stress, and relies on protein–protein interactions establishing a PML protein meshwork capable of recruiting the tumor suppressor p53, along with numerous modifiers of p53, thus balancing p53 posttranslational modifications and activity. This stress-regulated process appears to be controlled by liquid–liquid phase separation (LLPS), which may facilitate regulated protein-unmixing of p53 and its regulators into PML nuclear condensates. In this review, we summarize and discuss the molecular mechanisms underlying PML nuclear condensate formation, and how these impact the biological function of p53 in driving the cell death and senescence responses. In addition, by using an in silico approach, we identify 299 proteins which share PML and p53 as binding partners, thus representing novel candidate proteins controlling p53 function and cell fate decision-making at the level of PML nuclear biocondensates.

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