Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication

Boeckemeier, L.; Kraehenbuehl, Rolf; Keszthelyi, Andrea; Gasasira, M. U.; Vernon, Ellen; Beardmore, Richard; Vågbø, Cathrine Broberg; Chaplin, Daniel; Gollins, Simon; Krokan, Hans E.; Lambert, Sarah; Paizs, Béla; Hartsuiker, Edgar

doi:10.1126/sciadv.aaz4126

Cited by 10 publications

(10 citation statements)

References 58 publications

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“…Unlike TMZ, for a cell to resist treatment with dFdC (one active metabolite of gemcitabine), the chain-terminating nucleoside analog must be removed to allow replication restart [ 29 ]. However, the main HR repair factors such as ATM [ 30 ], RAD50 [ 30 ] or MRE11 [ 31 ], have been reported to remove dFdC but not increase HR repair efficiency. In our study of TMZ-resistance, HR efficiency was enhanced depended on ROCK2 induced ATM overexpression.…”

Section: Discussionmentioning

confidence: 99%

Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2

Zhang

Yang

et al. 2022

Cell Death Dis

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It was reported that MGMTlow gliomas may still be resistant to TMZ, while the mechanisms remain poorly understood. In this study, we demonstrated that rho-associated kinase 2 (ROCK2), a cytoskeleton regulator, was highly expressed in MGMTlow recurrent gliomas, and its expression strongly correlated with poor overall survival (OS) time in a subset of MGMTlow recurrent gliomas patients with TMZ therapy. And we also found that overactive ROCK2 enhanced homologous recombination repair (HR) in TMZ-resistant (TMZ-R) glioma cell lines with low MGMT expression. Silencing ROCK2 impaired HR repair, and induced double-strand break (DSB) and eradicated TMZ-R glioma cells in culture. Notably, in MGMTlow TMZ-R models, as a key factor of HR, ataxia telangiectasia-mutated (ATM) expression was upregulated directly by hyper-activation of ROCK2 to improve HR efficiency. ROCK2 enhanced the binding of transcription factor zinc finger E-box binding homeobox 1 (ZEB1) to ATM promoter for increasing ATM expression. Moreover, ROCK2 transformed ZEB1 into a gene activator via Yes-associated protein 1 (YAP1). These results provide evidence for the use of ROCK inhibitors in the clinical therapy for MGMTlow TMZ-resistant glioma. Our study also offered novel insights for improving therapeutic management of MGMTlow gliomas.

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Section: Discussionmentioning

confidence: 99%

Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2

Zhang

Yang

et al. 2022

Cell Death Dis

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“…While previous studies have attributed specific DNA damage repair pathways as defining therapeutic responses to replication stress-inducing nucleoside analogs 33,34,[67][68][69][70] , the exact mechanism(s) that underlie impaired resolution of cytarabine-induced DNA lesions in cells with mutant DNMT3A remain uncertain. Interestingly, other than a slight upregulation of genes annotated to be repressed during UV response indicative of a possible disruption of nucleotide excision repair 42 (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Venugopal

Nowialis

Feng

et al. 2021

Preprint

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Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

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“…Such distinctive sensitivity to cytarabine arises following a defect in recovery from replication fork arrest that leads to accumulation of persistent, unrepaired DNA damage observed in cells with mutant DNMT3A. While previous studies have attributed specific DNA damage repair pathways as defining therapeutic responses to replication stress-inducing nucleoside analogs (28,(56)(57)(58)(59), the exact mechanism(s) that underlie impaired resolution of cytarabine-induced DNA lesions in cells with mutant DNMT3A remain uncertain. Interestingly, other than a slight upregulation of genes annotated to be repressed during UV response indicative of a possible disruption of nucleotide excision repair (34) (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Venugopal

Feng

Nowialis

et al. 2021

Clinical Cancer Research

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Purpose: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Experimental Design: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. Results: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Conclusions: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573

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Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication

Cited by 10 publications

References 58 publications

Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2

Acquired temozolomide resistance in MGMTlow gliomas is associated with regulation of homologous recombination repair by ROCK2

DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

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