Targeting the DNA replication stress phenotype of KRAS mutant cancer cells

AbstractMutant KRAS is a common tumor driver and frequently confers resistance to anti-cancer treatments such as radiation. DNA replication stress in these tumors may constitute a therapeutic liability but is poorly understood. Here, using single-molecule DNA fiber analysis, we first characterized baseline replication stress in a panel of unperturbed isogenic and non-isogenic cancer cell lines. Correlating with the observed enhanced replication stress we found increased levels of cytosolic double-stranded DNA in KRAS mutant compared to wild-type cells. Yet, despite this phenotype replication stress-inducing agents failed to selectively impact KRAS mutant cells, which were protected by CHK1. Similarly, most exogenous stressors studied did not differentially augment cytosolic DNA accumulation in KRAS mutant compared to wild-type cells. However, we found that proton radiation was able to slow fork progression and preferentially induce fork stalling in KRAS mutant cells. Proton treatment also partly reversed the radioresistance associated with mutant KRAS. The cellular effects of protons in the presence of KRAS mutation clearly contrasted that of other drugs affecting replication, highlighting the unique nature of the underlying DNA damage caused by protons. Taken together, our findings provide insight into the replication stress response associated with mutated KRAS, which may ultimately yield novel therapeutic opportunities.

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DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status

10.1101/764522 ◽

2019 ◽

Author(s):

Yuki Kataoka ◽

Makoto Iimori ◽

Ryo Fujisawa ◽

Tomomi Morikawa-Ichinose ◽

Shinichiro Niimi ◽

...

Keyword(s):

Dna Replication ◽

Tumor Cell ◽

Tumor Cells ◽

Cell Fate ◽

Replication Fork ◽

Apoptotic Cell ◽

Replication Stress ◽

Dna Replication Stress ◽

P53 Status ◽

Fork Stalling

ABSTRACTDNA replication stress is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analog-type chemotherapeutic drugs introduce DNA damage and exacerbate DNA replication stress in tumor cells. However, the mechanisms underlying tumor cytotoxicity triggered by the drugs are not fully understood. Here, we show that the fluorinated thymidine analog trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases. FTD acted as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand. At the cellular level, FTD decreased thymidine triphosphate in the dNTP pool and induced FTD triphosphate accumulation, resulting in replication fork stalling caused by FTD incorporation into DNA. DNA lesions involving single-stranded DNA were generated as a result of replication fork stalling, and the p53-p21 pathway was activated. Although FTD suppressed tumor cell growth irrespective of p53 status, tumor cell fate diverged at the G2/M phase transition according to p53 status; tumor cells with wild-type p53 underwent cellular senescence via mitosis skip, whereas tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results suggest that DNA replication stress induced by a nucleoside analog-type chemotherapeutic drug triggers tumor cytotoxicity by determining tumor cell fate according to p53 status.SignificanceThis study identified a unique type of DNA replication stress induced by trifluridine, which directs tumor cell fate either toward cellular senescence or apoptotic cell death according to p53 status.

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Chromatin as a Platform for Modulating the Replication Stress Response

Genes ◽

10.3390/genes9120622 ◽

2018 ◽

Vol 9 (12) ◽

pp. 622 ◽

Cited By ~ 6

Author(s):

Louis-Alexandre Fournier ◽

Arun Kumar ◽

Peter Stirling

Keyword(s):

Dna Replication ◽

Stress Response ◽

Replication Fork ◽

Replication Stress ◽

Genome Maintenance ◽

Post Translational Modifications ◽

Nucleosome Dynamics ◽

Dna Replication Stress ◽

Eukaryotic Dna ◽

Fork Stalling

Eukaryotic DNA replication occurs in the context of chromatin. Recent years have seen major advances in our understanding of histone supply, histone recycling and nascent histone incorporation during replication. Furthermore, much is now known about the roles of histone remodellers and post-translational modifications in replication. It has also become clear that nucleosome dynamics during replication play critical roles in genome maintenance and that chromatin modifiers are important for preventing DNA replication stress. An understanding of how cells deploy specific nucleosome modifiers, chaperones and remodellers directly at sites of replication fork stalling has been building more slowly. Here we will specifically discuss recent advances in understanding how chromatin composition contribute to replication fork stability and restart.

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A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2109334118 ◽

2021 ◽

Vol 118 (38) ◽

pp. e2109334118

Author(s):

Albert Serra-Cardona ◽

Chuanhe Yu ◽

Xinmin Zhang ◽

Xu Hua ◽

Yuan Yao ◽

...

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Replication Stress ◽

Replication Checkpoint ◽

Checkpoint Pathway ◽

Dna Replication Checkpoint ◽

Dna Replication Stress ◽

Leading Strand ◽

Mutant Cells ◽

Lagging Strand

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

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p53 Reaction to Cellular Stress Maximizes Apoptosis in Glioblastoma

10.20944/preprints201901.0315.v1 ◽

2019 ◽

Author(s):

Cheng-Jung Ho ◽

Yu-Hsuan Pao ◽

Hsin-Wen Chen ◽

Wei-Hua Zhu ◽

Ru-Wei Lin ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Synergistic Effect ◽

Cellular Stress ◽

High Stress ◽

Replication Stress ◽

26S Proteasome ◽

Wild Type ◽

Apoptotic Response ◽

Dna Replication Stress

In prostate cancer, p53 maximizes apoptosis in response to severe DNA damage, not DNA replication stress. Here, we examined the apoptotic response of two glioblastoma cells, p53-wild type U87 and a p53-mutated T98G cell, for the same stresses. We ascertained that p53 intensified apoptosis in response to severe DNA damage, not DNA replication stress in glioblastoma. We further asked if p53-mediated apoptosis can be induced by cellular stress other than severe DNA damage. We analyzed two compounds, bortezomib and vorinostat, respective inhibitors of 26S proteasome and histone deacetylase, to evaluate their capacity to activate p53-mediated apoptosis. The cellular stress incited by bortezomib, not vorinostat, activated p53-mediated apoptosis. Next, we asked if the cellular stress generated by combining the two compounds had a synergistic effect on apoptosis. Our results demonstrated that doxorubicin with bortezomib or CFS-1686, or bortezomib with vorinostat have a significant synergistic effect on apoptosis only in p53-wild type cell. Under high stress, p53 translocates from cytosol into the nucleus to cause apoptosis possibly. Together, p53 maximizes apoptosis for cellular stress caused by severe DNA damage, disruption of protein turnover, and for the stress induced by drug combination including doxorubicin with bortezomib or CFS-1686, and bortezomib with vorinostat.

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