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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Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication

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 2

GOF p53 activates intra–S phase checkpoint, preventing collapse of replication forks.

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GOF p53 activates intra–S phase checkpoint, preventing collapse of repli...
Detection of p-CHK1, CHK1, and chromatin-associated γH2AX by immunoblot analysis using (A) lung cells of p53–/– and p53R172H-KI (R172H) mice and (C) mock-depleted (shGFP) or p53-depleted (shp53) H1048 lung cancer cells. For detection of chromatin-associated γH2AX, insoluble chromatin fraction was acid extracted and analyzed by immunoblotting. Band intensities of γH2AX normalized by H3 levels are shown by bar graphs (B and D). Lung cells from 2 mice of each construct or 2 clones stably expressing shGFP or shp53 were analyzed. The experimental strategy for detection of replication fork collapse by fiber analysis is shown by a schematic (E). Collapse of progressing replication forks was assayed by fiber analysis of replicating DNA. The bar graph (F) compares the percentage of red-only tracks (indicated by arrows in the fiber images) in replicating fibers. Approximately 200 untangled fibers from each sample were scored. The P value calculated using Student’s t test is indicated at the bottom of the bar graph (F). Lung cells from 2 mice of each construct were analyzed. Chromatin-associated γH2AX levels in lung cells from p53–/– and R172H mice after treatment with a CHK1 inhibitor, PF477736, were determined by immunoblot analysis (G). γH2AX band intensities normalized by H3 levels are shown by bar graphs (H). p-RPA (Ser4/Ser8) was detected by immunoblot analysis (G) to ensure activity of PF477736. Each experiment was repeated 3 times, and representative data are shown.