Loss-of-Function but Not Gain-of-Function Properties of Mutant TP53 Are Critical for the Proliferation, Survival, and Metastasis of a Broad Range of Cancer Cells.

Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer-derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. SIGNIFICANCE: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201.

PLK1 promotes the mitotic surveillance pathway by controlling cytosolic 53BP1 availability.

53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactors p53 and USP28, it is part of the mitotic surveillance (or mitotic stopwatch) pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. Here, we show that Polo-like kinase 1 (PLK1) activity is essential for the time-dependent release of 53BP1 from kinetochores. PLK1 inhibition, which leads to 53BP1 persistence at kinetochores, prevents cytosolic 53BP1 association with p53 and results in a blunted MSP. Strikingly, the identification of CENP-F as the kinetochore docking partner of 53BP1 enabled us to show that measurement of mitotic timing by the MSP does not take place at kinetochores, as perturbing CENP-F-53BP1 binding had no measurable impact on the MSP. Taken together, we propose that PLK1 supports the MSP by generating a cytosolic pool of 53BP1 and that an unknown cytosolic mechanism enables the measurement of mitotic duration.

The PIDDosome: centrosome guardian and backup on the DNA damage response.

The PIDDosome is a Caspase-2-activating platform assembling in response to centrosome amplification or genotoxic stress. We have recently shown that both stimuli depend on ANKRD26 (ankyrin repeat domain-containing protein 26)-mediated localization of PIDD1 (p53-inducible protein with death domain) at the centrosome, demonstrating how this organelle can directly influence cell fate.

CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells.

hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).

Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.

Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.