Loss of p53 function at late stages of tumorigenesis confers ARF-dependent vulnerability to p53 reactivation therapy.

Cancer development is driven by activated oncogenes and loss of tumor suppressors. While oncogene inhibitors have entered routine clinical practice, tumor suppressor reactivation therapy remains to be established. For the most frequently inactivated tumor suppressor p53, genetic mouse models have demonstrated regression of p53-null tumors upon p53 reactivation. While this was shown in tumor models driven by p53 loss as the initiating lesion, many human tumors initially develop in the presence of wild-type p53, acquire aberrations in the p53 pathway to bypass p53-mediated tumor suppression, and inactivate p53 itself only at later stages during metastatic progression or therapy. To explore the efficacy of p53 reactivation in this scenario, we used a reversibly switchable p53 (p53ERTAM) mouse allele to generate Eµ-Myc-driven lymphomas in the presence of active p53 and, after full lymphoma establishment, switched off p53 to model late-stage p53 inactivation. Although these lymphomas had evolved in the presence of active p53, later loss and subsequent p53 reactivation surprisingly activated p53 target genes triggering massive apoptosis, tumor regression, and long-term cure of the majority of animals. Mechanistically, the reactivation response was dependent on Cdkn2a/p19Arf, which is commonly silenced in p53 wild-type lymphomas, but became reexpressed upon late-stage p53 inactivation. Likewise, human p53 wild-type tumor cells with CRISPR-engineered switchable p53ERTAM alleles responded to p53 reactivation when CDKN2A/p14ARF function was restored or mimicked with Mdm2 inhibitors. Together, these experiments provide genetic proof of concept that tumors can respond, in an ARF-dependent manner, to p53 reactivation even if p53 inactivation has occurred late during tumor evolution.

Phosphorylation Control of p53 DNA-Binding Cooperativity Balances Tumorigenesis and Aging.

Posttranslational modifications are essential for regulating the transcription factor p53, which binds DNA in a highly cooperative manner to control expression of a plethora of tumor-suppressive programs. Here we show at the biochemical, cellular, and organismal level that the cooperative nature of DNA binding is reduced by phosphorylation of highly conserved serine residues (human S183/S185, mouse S180) in the DNA-binding domain. To explore the role of this inhibitory phosphorylation in vivo, new phosphorylation-deficient p53-S180A knock-in mice were generated. Chromatin immunoprecipitation sequencing and RNA sequencing studies of S180A knock-in cells demonstrated enhanced DNA binding and increased target gene expression. In vivo, this translated into a tissue-specific vulnerability of the bone marrow that caused depletion of hematopoietic stem cells and impaired proper regeneration of hematopoiesis after DNA damage. Median lifespan was significantly reduced by 20% from 709 days in wild type to only 568 days in S180A littermates. Importantly, lifespan was reduced by a loss of general fitness and increased susceptibility to age-related diseases, not by increased cancer incidence as often seen in other p53-mutant mouse models. For example, S180A knock-in mice showed markedly reduced spontaneous tumorigenesis and increased resistance to Myc-driven lymphoma and Eml4-Alk-driven lung cancer. Preventing phosphorylation of S183/S185 in human cells boosted p53 activity and allowed tumor cells to be killed more efficiently. Together, our data identify p53 DNA-binding domain phosphorylation as a druggable mechanism that balances tumorigenesis and aging. SIGNIFICANCE: These findings demonstrate that p53 tumor suppressor activity is reduced by DNA-binding domain phosphorylation to prevent aging and identify this phosphorylation as a potential target for cancer therapy.See related commentary by Horikawa, p. 5164.