Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication.

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.

Constitutive Activation of DNA Damage Checkpoint Signaling Contributes to Mutant p53 Accumulation via Modulation of p53 Ubiquitination.

UNLABELLED: Many mutant p53 proteins exhibit an abnormally long half-life and overall increased abundance compared with wild-type p53 in tumors, contributing to mutant p53's gain-of-function oncogenic properties. Here, a novel mechanism is revealed for the maintenance of mutant p53 abundance in cancer that is dependent on DNA damage checkpoint activation. High-level mutant p53 expression in lung cancer cells was associated with preferential p53 monoubiquitination versus polyubiquitination, suggesting a role for the ubiquitin/proteasome system in regulation of mutant p53 abundance in cancer cells. Interestingly, mutant p53 ubiquitination status was regulated by ataxia-telangectasia mutated (ATM) activation and downstream phosphorylation of mutant p53 (serine 15), both in resting and in genotoxin-treated lung cancer cells. Specifically, either inhibition of ATM with caffeine or mutation of p53 (serine 15 to alanine) restored MDM2-dependent polyubiquitination of otherwise monoubiquitinated mutant p53. Caffeine treatment rescued MDM2-dependent proteasome degradation of mutant p53 in cells exhibiting active DNA damage signaling, and ATM knockdown phenocopied the caffeine effect. Importantly, in cells analyzed individually by flow cytometry, p53 levels were highest in cells exhibiting the greatest levels of DNA damage response, and interference with DNA damage signaling preferentially decreased the relative percentage of cells in a population with the highest levels of mutant p53. These data demonstrate that active DNA damage signaling contributes to high levels of mutant p53 via modulation of ubiquitin/proteasome activity toward p53. IMPLICATION: The ability of DNA damage checkpoint signaling to mediate accumulation of mutant p53 suggests that targeting this signaling pathway may provide therapeutic gain. Mol Cancer Res; 14(5); 423-36. ©2016 AACR.

Mechanisms of mutant p53 stabilization in cancer.

p53 transactivates cell cycle inhibitory, apoptosis or senescence-related genes in response to DNA damage to protect the genetic integrity of the cell. Highlighting its critical tumor suppressor functions, p53 is mutated, lost, or functionally inactivated in nearly all cancers. When mutated within its core DNA binding domain, p53's normal instability is abrogated, and oncogenic gain-of-function properties are observed accompanied by massive accumulation of steady state mutant p53 protein levels relative to the low or undetectable steady state level of wild-type (WT) p53 in normal cells. Mutation of p53 may affect its stability through a combination of mutant p53's inherent biochemical and biophysical properties as well as pathways aberrantly activated in genetically damaged cells. The increased stability of mutant p53 proteins is key to its ability to accumulate to high levels and phenotypically exhibit "gain-of-function" properties. In this chapter we will address the multifaceted ways in which intrinsic mutant p53 properties intersect with emergent properties of cancer cells to yield the stable mutant p53 phenotype.

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing.

Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53-/-:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53-/- mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.

Use of the DNA fiber spreading technique to detect the effects of mutant p53 on DNA replication.

DNA replication involves a coordinated progression through S phase, and disruption of these regulated steps may cause gene abnormalities, which may lead to cancer. Different stages of DNA replication can be detected immunofluorescently that would indicate how replication is progressing in a cell population or under specific conditions. We describe a method for labeling replicating DNA with two nucleotide analogs, and then detecting the sequential patterns of incorporation using fluorescently labeled antibodies on DNA spread onto a glass slide. Quantification of the different types of replication patterns produced by this method reveals how replication is achieved under different conditions by the predominance and lengths of elongating replication forks progressing from single or clustered origins, as well as the sites of termination from two converging forks.

Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells.

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.

DNA replication in early S phase pauses near newly activated origins.

During the S phase of the cell cycle, the entire genome is replicated. There is a high level of orderliness to this process through the temporally and topologically coordinated activation of many replication origins situated along chromosomes. We investigated the program of replication from origins initiating in early S phase by labeling synchronized normal human fibroblasts (NHF1) with nucleotide analogs for various pulse times and measuring labeled tracks in combed DNA fibers. Our analysis showed that replication forks progress 9-35 kilobases from newly initiated origins, followed by a pause in synthesis before replication resumes. Pausing was not observed near origins that initiated in the middle of S phase. No evidence for pausing near origins was found at the beginning of the S phase in glioblastoma T98G cells. Treatment with the S phase checkpoint inhibitor caffeine abrogated pausing in NHF1 cells in early S phase. This suggests that pausing may comprise a novel aspect of the intra-S phase checkpoint pathway or a related new early S checkpoint. Further, it is possible that the loss of this regulatory process in cancer cells such as T98G could be a contributing factor in the genetic instability that typifies cancers.