p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.

Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9–based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.

The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73γ have basic CTDs and form weak sequence-specific protein–DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein–DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73).

Background: The efficacy of PD-(L)1 blockade depends on the composition of the tumor immune microenvironment (TIME) and is generally higher in tumors with pre-existing cytotoxic T cells (CTL) than in those with low CTL numbers. Nonetheless, a significant proportion of patients with pre-existing immunity fail to respond, indicating a therapeutic potential for combining PD-(L)1 blockade with additional immunomodulatory agents in both CTL-high and-low immune phenotypes. Here, we evaluated domatinostat (4SC-202), a class I-selective histone deacetylase (HDAC) inhibitor, for its effect on the TIME and its antitumoral efficacy using syngeneic mouse models with CTL-high or CTL-low tumors. Methods: Domatinostat was evaluated in PD-1 blockade-insensitive CTL-low (CT26) and CTL-high (C38) syngeneic models alone and in combination with different immune-inhibitory and-stimulatory approaches. Effects on the immunophenotype were assessed via flow cytometry and RNA-seq analyses. The changes in RNA-seq-based immune signatures determined in a murine setting were investigated in patient samples from the first-dose cohort of the SENSITIZE trial (NCT03278665) evaluating domatinostat combined with pembrolizumab in advanced-stage melanoma patients refractory/nonresponding to PD-1 blockade. Results: Domatinostat increased the expression of antigen-presenting machinery (APM) genes and MHC class I and II molecules, along with CTL infiltration, in tumors of both immune phenotypes. In combination with PD-(L)1 blockade, domatinostat augmented antitumor effects substantially above the effects of single-agent therapies, displaying greater benefit in tumors with pre-existing CTLs. In this setting, the combination of domatinostat with agonistic anti-4-1BB or both PD-1 and LAG3 blockade further increased the antitumor efficacy. In CTL-low tumors, domatinostat enhanced the expression of genes known to reinforce immune responses against tumors. Specifically, domatinostat increased the expression of Ifng and genes associated with responses to pembrolizumab and nivolumab. Clinically, these findings were confirmed in patients with advanced melanoma treated with domatinostat for 14 days, who demonstrated elevated expression of APM and MHC genes, the IFNG gene, and the IFN-γ and pembrolizumab response signatures in individual tumor samples. Conclusion: In summary, these data suggest a promising potential of domatinostat in combination with immunotherapy to improve the outcome of refractory cancer patients.

p53 is known to prevent tumour formation by restricting the proliferation of damaged or oncogene-expressing cells. In contrast, how the p53 family member p73 suppresses tumour formation remains elusive. Using a step-wise transformation protocol for human cells, we show that, in premalignant stages, expression of the transactivationcompetent p73 isoform TAp73 is triggered in response to pRB pathway alterations. TAp73 expression at this stage of transformation results in increased sensitivity to chemotherapeutic drugs and oxidative stress and inhibits proliferation and survival at high cell density. Importantly, TAp73 triggers a transcriptional programme to prevent anchorage-independent growth, which is considered a crucial hallmark of fully transformed cells. An essential suppressor of anchorage-independent growth is KCNK1, which is directly transactivated by TAp73 and commonly downregulated in glioma, melanoma and ovarian cancer. Oncogenic Ras switches p73 expression from TAp73 to the oncogenic DNp73 isoform in a phosphatidyl-inositol 3-kinase-dependent manner. Our results implicate TAp73 as a barrier to anchorage-independent growth and indicate that downregulation of TAp73 is a key transforming activity of oncogenic Ras mutants.