Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.

The tumor suppressive transcription factor p53 is frequently inactivated in cancer cells by missense mutations that cluster in the DNA binding domain. 30% hit mutational hotspot residues, resulting in a complete loss of transcriptional activity and mutant p53-driven chemotherapy resistance. Of the remaining 70% of non-hotspot mutants, many are partial loss-of-function (partial-LOF) mutants with residual transcriptional activity. The therapeutic consequences of a partial-LOF have remained largely elusive. Using a p53 mutation engineered to reduce DNA binding, we demonstrate that partial-LOF is sufficient to enhance oncogene-driven tumorigenesis in mouse models of lung and pancreatic ductal adenocarcinoma and acute myeloid leukemia. Interestingly, mouse and human tumors with partial-LOF mutations showed mutant p53 protein accumulation similar as known for hotspot mutants. Different from the chemotherapy resistance caused by p53-loss, the partial-LOF mutant sensitized to an apoptotic chemotherapy response and led to a survival benefit. Mechanistically, the pro-apoptotic transcriptional activity of mouse and human partial-LOF mutants was rescued at high mutant protein levels, suggesting that accumulation of partial-LOF mutants enables the observed apoptotic chemotherapy response. p53 non-hotspot mutants with partial-LOF, therefore, represent tumorigenic p53 mutations that need to be distinguished from other mutations because of their beneficial impact on survival in a therapy context.

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

Background: Impaired p53 function is one of the central molecular features of a tumor cell and even a partial reduction in p53 activity can increase the cancer risk in mice and men. From a therapeutic perspective it is noteworthy that tumor cells often become addicted to the absence of p53 providing a rationale for developing p53 reactivating compounds to treat cancer patients. Unfortunately, many of the compounds that are currently undergoing preclinical and clinical testing fail to fully reactivate mutant p53 proteins, raising the crucial question: how much p53 activity is needed to elicit a therapeutic effect? Methods: We have genetically modelled partial p53 reactivation using knock-in mice with inducible expression of the p53 variant E177R. This variant has a reduced ability to bind and transactivate target genes and consequently causes moderate cancer susceptibility. We have generated different syngeneically transplanted and autochthonous mouse models of p53-deficient acute myeloid leukemia and B or T cell lymphoma. After cancer manifestation we have activated E177R expression and analyzed the in vivo therapy response by bioluminescence or magnetic resonance imaging. The molecular response was further characterized in vitro by assays for gene expression, proliferation, senescence, differentiation, apoptosis and clonogenic growth. Results: We report the conceptually intriguing observation that the p53 variant E177R, which promotes de novo leukemia and lymphoma formation, inhibits proliferation and viability, induces immune cell infiltration and triggers cancer regression in vivo when introduced into p53-deficient leukemia and lymphomas. p53-deficient cancer cells proved to be so addicted to the absence of p53 that even the low-level activity of E177R is detrimental to cancer growth. Conclusions: The observation that a partial loss-of-function p53 variant promotes tumorigenesis in one setting and induces regression in another, underlines the highly context-specific effects of individual p53 mutants. It further highlights the exquisite sensitivity of cancer cells to even small changes in p53 activity and reveals that changes in activity level are more important than the absolute level. As such, the study encourages ongoing research efforts into mutant p53 reactivating drugs by providing genetic proof-of-principle evidence that incomplete p53 reactivation may suffice to elicit a therapeutic response.

P53 is a tumor suppressor that is mutated in half of all cancers. The high clinical relevance has made p53 a model transcription factor for delineating general mechanisms of transcriptional regulation. p53 forms tetramers that bind DNA in a highly cooperative manner. The DNA binding cooperativity of p53 has been studied by structural and molecular biologists as well as clinical oncologists. These experiments have revealed the structural basis for cooperative DNA binding and its impact on sequence specificity and target gene spectrum. Cooperativity was found to be critical for the control of p53-mediated cell fate decisions and tumor suppression. Importantly, an estimated number of 34,000 cancer patients per year world-wide have mutations of the amino acids mediating cooperativity, and knock-in mouse models have confirmed such mutations to be tumorigenic. While p53 cancer mutations are classically subdivided into “contact” and “structural” mutations, “cooperativity” mutations form a mechanistically distinct third class that affect the quaternary structure but leave DNA contacting residues and the three-dimensional folding of the DNA-binding domain intact. In this review we discuss the concept of DNA binding cooperativity and highlight the unique nature of cooperativity mutations and their clinical implications for cancer therapy.

TP53 mutations are found in 50% of all cancers and mutated TP53 status is considered poor for treatment. However, some TP53 mutations exhibit only partial loss-of-function (LOF), meaning they retain residual transcriptional and non-transcriptional activities that are potentially beneficial for therapy. Earlier we have characterized a knock-in mouse model for the partial LOF mutant Trp53^E177R (p53RR). Reduced DNA binding cooperativity of this mutant led to the loss of p53-dependent apoptosis, while p53 functions in cell cycle control, senescence, metabolism, and antioxidant defense remained intact. Concomitantly, tumor suppression was evident but strongly compromised compared to wild-type mice. Here we used the Trp53^E177R mouse as a model to investigate whether residual functions of mutant p53 can be engaged to induce cell death, which is considered the most desirable outcome of tumor therapy. We made use of Mdm2 knock-out in developing embryos as a sensitive tool for detecting remaining p53 activities. Genetic ablation of Mdm2 led to embryonic lethality in Trp53^E177R/E177R homozygotes at days 9.5–11.5. This effect was not rescued by concomitant p21-knockout, indicating its independence of p21-mediated cell cycle arrest. Instead, immunohistochemical analysis showed widespread apoptosis in tissues of defective embryos accompanied by persistent accumulation of p53RR protein. This led to partial restoration of the mutant’s proficiency in transcriptional induction of the pro-apoptotic genes Bbc3 (Puma) and Bax. These data indicate that increased quantity can compensate for qualitative defects of p53 mutants and suggest that Mdm2-targeting (potentially in combination with other drugs) might be effective against cells bearing p53 partial LOF mutants.