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Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication
Shilpa Singh, … , Sumitra Deb, Swati Palit Deb
Shilpa Singh, … , Sumitra Deb, Swati Palit Deb
Published April 10, 2017
Citation Information: J Clin Invest. 2017;127(5):1839-1855. https://doi.org/10.1172/JCI87724.
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Research Article Cell biology Oncology Article has an altmetric score of 48

Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication

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Abstract

Gain-of-function (GOF) p53 mutations are observed frequently in most intractable human cancers and establish dependency for tumor maintenance and progression. While some of the genes induced by GOF p53 have been implicated in more rapid cell proliferation compared with p53-null cancer cells, the mechanism for dependency of tumor growth on mutant p53 is unknown. This report reveals a therapeutically targetable mechanism for GOF p53 dependency. We have shown that GOF p53 increases DNA replication origin firing, stabilizes replication forks, and promotes micronuclei formation, thus facilitating the proliferation of cells with genomic abnormalities. In contrast, absence or depletion of GOF p53 leads to decreased origin firing and a higher frequency of fork collapse in isogenic cells, explaining their poorer proliferation rate. Following genome-wide analyses utilizing ChIP-Seq and RNA-Seq, GOF p53–induced origin firing, micronuclei formation, and fork protection were traced to the ability of GOF p53 to transactivate cyclin A and CHK1. Highlighting the therapeutic potential of CHK1’s role in GOF p53 dependency, experiments in cell culture and mouse xenografts demonstrated that inhibition of CHK1 selectively blocked proliferation of cells and tumors expressing GOF p53. Our data suggest the possibility that checkpoint inhibitors could efficiently and selectively target cancers expressing GOF p53 alleles.

Authors

Shilpa Singh, Catherine A. Vaughan, Rebecca A. Frum, Steven R. Grossman, Sumitra Deb, Swati Palit Deb

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Figure 5

The transactivation function of GOF p53 is required for cell proliferation, increased frequency of replication origin firing, micronuclei formation, CHK1 expression and phosphorylation, and replication fork protection.

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The transactivation function of GOF p53 is required for cell proliferati...
(A) Schematic showing functional domains of p53, including its TADI and TADII, sites of hot spot mutations, and created mutations on TADI (L22Q and W23S) and TADII (L53Q and F54S). Reg, regulatory. (B) Rate of proliferation of H1299 cells stably expressing p53R273H or its TADI mutant was determined. Data are presented as mean ± SD of triplicate experiments. Two clones of TADI mutants were used. (C) p53 expression was confirmed by immunoblot analysis. (D) Origin firing was determined by fiber analysis of replicating DNA. Data obtained by scoring 200 untangled fibers are shown by a bar graph. P value calculated using Student’s t test is shown at the bottom of the graph. (E) Micronuclei formation was determined by DAPI staining. Data obtained by scoring 500 cells are shown by bar graphs. P value calculated using χ2 test is shown at the bottom of the bar graphs. (F) Expression of CHK1 and p-CHK1 (at S345) in H1299 cells expressing p53R273H or its TADI mutant was determined by immunoblot analysis. (G) γH2AX foci formation in H1299 cells expressing p53R273H or TADI mutant was determined by immunostaining with Alexa Fluor 488–coupled γH2AX antibody followed by confocal microscopy. Arrows indicate nuclei with green foci. Original magnification: ×20. (H) Frequency of nuclei with γH2AX foci in each construct is shown by bar graph; 250 cells were scored, and P value was calculated using Student’s t test. Each experiment was repeated 3 times.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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