Sabrina Elmshäuser

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 mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.

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.

Abstract 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

Abstract Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53 R178E mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53 −/− mice. Surprisingly, stabilization of p53 R178E in Mdm2 −/− mice nevertheless triggers extensive apoptosis, indicative of residual wild‐type activities. Although this apoptotic activity suffices to trigger lethality of Trp53 R178E ;Mdm2 −/− embryos, it proves insufficient for suppression of spontaneous and oncogene‐driven tumorigenesis. Trp53 R178E mice develop tumors indistinguishably from Trp53 −/− mice and tumors retain and even stabilize the p53 R178E protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53 R178E tumors exhibit remarkably better chemotherapy responses than Trp53 −/− ones, resulting in enhanced eradication of p53‐mutated tumor cells. Together, this provides genetic proof‐of‐principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy.