EXTH-05. THERAPEUTIC IMPLICATIONS OF TTFIELDS INDUCED DNA DAMAGE AND REPLICATION STRESS IN NOVEL COMBINATIONS FOR CANCER TREATMENT

Abstract TTFields are low-intensity, intermediate frequency, alternating electric fields which are applied to tumor regions using non-invasive arrays. TTFields is approved for the treatment of glioblastoma and mesothelioma with clinical trials ongoing in other cancer types. The mechanism of action for TTFields includes interference with mitosis, reduced DNA double strand break (DSB) repair capacity and the frank induction of DNA DSBs. The mechanism by which TTFields induces DNA DSBs appears to be through the enhancement of DNA replication stress with continued TTFields exposure. The induction of DNA DSBs appears to be as a result of significantly reduced expression of the DNA replication complex genes MCM6 and MCM10 as well as the Fanconi’s Anemia (FA) pathway genes. TTFields treatment increases the number of RPA foci, decreases nascent DNA length and increases R-loop formation which are markers of DNA replication stress. These results suggest that TTFields-induced replication stress is the underlying mechanism and cellular endogenous source of DNA DSB generation via replication fork collapse. The current study suggests that TTFields exposure causes a conditional vulnerability environment that renders cells more susceptible to chemotherapeutic agents that induce DNA damage and/or cause replication stress. Supporting this is the synergistic cell killing seen with TTFields exposure concomitant with cisplatin, TTFields plus concomitant PARP inhibition with or without subsequent radiation, or radiation given at the completion of a TTFields exposure. Finally, TTFields-induced mitotic aberrations and DNA damage/replication stress events, although intimately linked to one another as one can expose the other, are likely initiated independently of one another as suggested by the gene expression analysis of 47 key mitosis regulator genes. These results establish that enhanced replication stress and reduced DNA repair capacity are also major mechanisms of TTFields effects, effects for which there are therapeutic implications.

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MBRS-71. ATAXIA TELANGIECTASIA AND RAD3-RELATED PROTEIN ATTENUATES DNA DAMAGE AND IS A THERAPEUTIC TARGET IN Myc-DRIVEN MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.575 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii411-iii411

Author(s):

Ahmed Abdel-Hafiz ◽

Krishna Madhavan ◽

Ilango Balakrishnan ◽

Angela Pierce ◽

Dong Wang ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Cell Lines ◽

Ataxia Telangiectasia ◽

Replication Stress ◽

Chemotherapeutic Agents ◽

Related Protein ◽

Dna Replication Stress ◽

Group 3 ◽

Orthotopic Xenografts

Abstract Group 3 medulloblastoma tumors (Myc-MB), and particularly the 3γ subtype, have the worst prognosis and show a 5-year overall survival of less than 40%. Group 3 tumors are often accompanied by Myc amplification and have a higher rate of metastatic disease and relapse. Unfortunately, therapeutic strategies to target Mychave remained elusive. Further, the relapse of the MB has been linked to DNA replication stress. Ataxia telangiectasia and Rad3-related protein (ATR) senses persistent DNA damage, which arises due to replication stress, and activates damage checkpoints, thereby leading to increased cell survival. ATR is highly expressed in MB and is thought to contribute to undisturbed DNA replication to protect genomic integrity. Yet, the exact underlying mechanisms involving ATR are still unclear in MB. Inhibition of ATR (ATRi) using the ATR inhibitor, AZD6738, suppressed clonogenicity and cell self-renewal in Myc-MB. ATRi in Myc-MB cell lines downregulated Chk1 and upregulated P21. ATRi also induced cell cycle arrest and increased apoptosis in Myc-MB cell lines. Further, mice with orthotopic xenografts treated with ATR inhibitor survived significantly longer than control mice. High-throughput drug screening showed ATRi to be synergistic with chemotherapeutic agents including gemcitabine, cisplatin and topotecan. The treatment of Myc-MB cells with ATR inhibitor in combination with gemcitabine and with radiation increased in expression of DNA damage markers. These findings emphasize the role of ATR in alleviating DNA replication stress and that its inhibition is critical to the treatment of Myc-MB.

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MTA2 sensitizes gastric cancer cells to PARP inhibition by induction of DNA replication stress

Translational Oncology ◽

10.1016/j.tranon.2021.101167 ◽

2021 ◽

Vol 14 (10) ◽

pp. 101167

Author(s):

Jinwen Shi ◽

Xiaofeng Zhang ◽

Jin'e Li ◽

Wenwen Huang ◽

Yini Wang ◽

...

Keyword(s):

Gastric Cancer ◽

Dna Replication ◽

Cancer Cells ◽

Replication Stress ◽

Parp Inhibition ◽

Gastric Cancer Cells ◽

Dna Replication Stress

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Protective Mechanisms Against DNA Replication Stress in the Nervous System

Genes ◽

10.3390/genes11070730 ◽

2020 ◽

Vol 11 (7) ◽

pp. 730

Author(s):

Clara Forrer Charlier ◽

Rodrigo A. P. Martins

Keyword(s):

Dna Damage ◽

Nervous System ◽

Dna Replication ◽

Neurological Diseases ◽

Replication Stress ◽

Nervous System Development ◽

Stress Generation ◽

Genome Replication ◽

Cns Development ◽

Dna Replication Stress

The precise replication of DNA and the successful segregation of chromosomes are essential for the faithful transmission of genetic information during the cell cycle. Alterations in the dynamics of genome replication, also referred to as DNA replication stress, may lead to DNA damage and, consequently, mutations and chromosomal rearrangements. Extensive research has revealed that DNA replication stress drives genome instability during tumorigenesis. Over decades, genetic studies of inherited syndromes have established a connection between the mutations in genes required for proper DNA repair/DNA damage responses and neurological diseases. It is becoming clear that both the prevention and the responses to replication stress are particularly important for nervous system development and function. The accurate regulation of cell proliferation is key for the expansion of progenitor pools during central nervous system (CNS) development, adult neurogenesis, and regeneration. Moreover, DNA replication stress in glial cells regulates CNS tumorigenesis and plays a role in neurodegenerative diseases such as ataxia telangiectasia (A-T). Here, we review how replication stress generation and replication stress response (RSR) contribute to the CNS development, homeostasis, and disease. Both cell-autonomous mechanisms, as well as the evidence of RSR-mediated alterations of the cellular microenvironment in the nervous system, were discussed.

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The deubiquitinase USP36 Regulates DNA replication stress and confers therapeutic resistance through PrimPol stabilization

Nucleic Acids Research ◽

10.1093/nar/gkaa1090 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12711-12726

Author(s):

Yuanliang Yan ◽

Zhijie Xu ◽

Jinzhou Huang ◽

Guijie Guo ◽

Ming Gao ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Stress Response ◽

Critical Role ◽

Replication Stress ◽

Regulatory Mechanisms ◽

Therapeutic Resistance ◽

Chemotherapy Response ◽

Dna Replication Stress ◽

Damage Tolerant

Abstract PrimPol has been recently identified as a DNA damage tolerant polymerase that plays an important role in replication stress response. However, the regulatory mechanisms of PrimPol are not well defined. In this study, we identify that the deubiquitinase USP36 interferes with degradation of PrimPol to regulate the replication stress response. Mechanistically, USP36 is deubiquitinated following DNA replication stress, which in turn facilitates its upregulation and interaction with PrimPol. USP36 deubiquitinates K29-linked polyubiquitination of PrimPol and increases its protein stability. Depletion of USP36 results in replication stress-related defects and elevates cell sensitivity to DNA-damage agents, such as cisplatin and olaparib. Moreover, USP36 expression positively correlates with the level of PrimPol protein and poor prognosis in patient samples. These findings indicate that the regulation of PrimPol K29-linked ubiquitination by USP36 plays a critical role in DNA replication stress and chemotherapy response.

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Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0639 ◽

2009 ◽

Vol 20 (3) ◽

pp. 983-994 ◽

Cited By ~ 13

Author(s):

Xia Yi ◽

Hilda I. de Vries ◽

Katarzyna Siudeja ◽

Anil Rana ◽

Willy Lemstra ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Synthesis ◽

Replication Stress ◽

Epigenetic Mechanism ◽

Dna Integrity ◽

Silent Chromatin ◽

Chromatin Compaction ◽

Cycle Arrest ◽

Dna Replication Stress

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.

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Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress

Nature Cell Biology ◽

10.1038/ncb2549 ◽

2012 ◽

Vol 14 (9) ◽

pp. 966-976 ◽

Cited By ~ 291

Author(s):

Johnny M. Tkach ◽

Askar Yimit ◽

Anna Y. Lee ◽

Michael Riffle ◽

Michael Costanzo ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Protein Localization ◽

Replication Stress ◽

Damage Response ◽

Dna Replication Stress

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The activation loop residue serine 173 of S.pombe Chk1 kinase is critical for the response to DNA replication stress

10.1101/164244 ◽

2017 ◽

Author(s):

Naomi Coulton ◽

Thomas Caspari

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Stress ◽

Genetic Alterations ◽

Activation Loop ◽

Replication Forks ◽

Dna Polymerase Delta ◽

Dna Replication Stress ◽

Summary Statement ◽

Chk1 Kinase

AbstractWhy the DNA damage checkpoint kinase Chk1 protects the genome of lower and higher eukaryotic cells differentially is still unclear. Mammalian Chk1 regulates replication origins, safeguards DNA replication forks and promotes fork progression. Conversely, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the activation loop of fission yeast Chk1 abolishes the G1-M and S-M checkpoints without affecting the G2-M arrest. Although Chk1-S173A is fully phosphorylated at serine 345 by the DNA damage sensor Rad3 (ATR) when DNA replication forks break, cells fail to stop the cell cycle. Mutant cells are uniquely sensitive to the DNA alkylation agent methyl- methanesulfate (MMS). This MMS sensitivity is genetically linked with the lagging strand DNA polymerase delta. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 is impaired. Serine 173 is equivalent to lysine 166 in human Chk1, an amino acid important for substrate specificity. We conclude that the removal of serine 173 impairs the phosphorylation of a Chk1 target that is important to protect cells from DNA replication stress.Summary statementMutation of serine-173 in the activation loop of Chk1 kinase may promote cancer as it abolishes the response to genetic alterations that arise while chromosomes are being copied.

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Centrosome impairment causes DNA replication stress through MLK3/MK2 signaling and R-loop formation

10.1101/2020.01.09.898684 ◽

2020 ◽

Author(s):

Zainab Tayeh ◽

Kim Stegmann ◽

Antonia Kleeberg ◽

Mascha Friedrich ◽

Josephine Ann Mun Yee Choo ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Replication Stress ◽

List Type ◽

Replication Forks ◽

Loop Formation ◽

Animal Cells ◽

Replication Fork Progression ◽

Primordial Dwarfism ◽

Dna Replication Stress

AbstractCentrosomes function as organizing centers of microtubules and support accurate mitosis in many animal cells. However, it remains to be explored whether and how centrosomes also facilitate the progression through different phases of the cell cycle. Here we show that impairing the composition of centrosomes, by depletion of centrosomal components or by inhibition of polo-like kinase 4 (PLK4), reduces the progression of DNA replication forks. This occurs even when the cell cycle is arrested before damaging the centrosomes, thus excluding mitotic failure as the source of replication stress. Mechanistically, the kinase MLK3 associates with centrosomes. When centrosomes are disintegrated, MLK3 activates the kinases p38 and MK2/MAPKAPK2. Transcription-dependent RNA:DNA hybrids (R-loops) are then causing DNA replication stress. Fibroblasts from patients with microcephalic primordial dwarfism (Seckel syndrome) harbouring defective centrosomes showed replication stress and diminished proliferation, which were each alleviated by inhibition of MK2. Thus, centrosomes not only facilitate mitosis, but their integrity is also supportive in DNA replication.HighlightsCentrosome defects cause replication stress independent of mitosis.MLK3, p38 and MK2 (alias MAPKAPK2) are signalling between centrosome defects and DNA replication stress through R-loop formation.Patient-derived cells with defective centrosomes display replication stress, whereas inhibition of MK2 restores their DNA replication fork progression and proliferation.Graphical abstract

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Harnessing DNA Replication Stress for Novel Cancer Therapy

Genes ◽

10.3390/genes11090990 ◽

2020 ◽

Vol 11 (9) ◽

pp. 990 ◽

Cited By ~ 2

Author(s):

Huanbo Zhu ◽

Umang Swami ◽

Ranjan Preet ◽

Jun Zhang

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Cancer Therapy ◽

Replication Stress ◽

Replicative Stress ◽

Combination Treatments ◽

Damage Repair ◽

Target Dna ◽

Dna Replication Stress ◽

Exogenous Factors

DNA replication is the fundamental process for accurate duplication and transfer of genetic information. Its fidelity is under constant stress from endogenous and exogenous factors which can cause perturbations that lead to DNA damage and defective replication. This can compromise genomic stability and integrity. Genomic instability is considered as one of the hallmarks of cancer. In normal cells, various checkpoints could either activate DNA repair or induce cell death/senescence. Cancer cells on the other hand potentiate DNA replicative stress, due to defective DNA damage repair mechanism and unchecked growth signaling. Though replicative stress can lead to mutagenesis and tumorigenesis, it can be harnessed paradoxically for cancer treatment. Herein, we review the mechanism and rationale to exploit replication stress for cancer therapy. We discuss both established and new approaches targeting DNA replication stress including chemotherapy, radiation, and small molecule inhibitors targeting pathways including ATR, Chk1, PARP, WEE1, MELK, NAE, TLK etc. Finally, we review combination treatments, biomarkers, and we suggest potential novel methods to target DNA replication stress to treat cancer.

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