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Histone deacetylase inhibitors target regulators of DNA replication and the cellular response to DNA replication stress in Ewing sarcoma cells

ABSTRACT: DNA replication stress is common feature of multiple tumor types. Histone deacetylases (HDAC) regulate diverse pathways in cancer cells and recent work has identified that HDAC inhibitors downregulate the expression of multiple proteins with critical roles in DNA replication and the cellular response to DNA replication stress, including the RRM1 and RRM2 subunits of ribonucleotide reductase (RNR), CHK1, and WEE1. RNA-seq analysis was performed on a Ewing sarcoma cell line to determine the effects of HDAC inhibitors on the transcriptome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE280573 | GEO | 2025/06/03

REPOSITORIES: GEO

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Identification of genes regulated by loss of RRM1 in Ewing sarcoma cells.

Project description:Ribonucleotide reductase (RNR), which is composed of RRM1 and RRM2 subunits, is the rate limiting enzyme in the synthesis of deoxyribonucleotides and required for DNA replication and DNA damage repair. We used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identify downstream pathways impacted by the loss of RNR activity. RNA-seq analysis was performed at times 0 and 48 hours after removal of doxycycline from cell culture media, which results in loss of the RRM1 protein.

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An essential role for dNTP homeostasis following CDKinduced [Pu-seq]

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Gene expression signature baesd screening identifies ribonucleotide reductase as a candidate therapeutic target in Ewing sarcoma

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Project description:The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinases Rad53 and Dun1. Partial disruption of the MCM/RNR interactions increases the levels of dNTPs under unperturbed conditions, pointing to a role for MCM counteracting excessive RNR activity. In response to DNA damage, most of these physical interactions augment and this increase depends on Dun1. Partial disruption of the MCM/RNR interactions does not affect the pool of dNTPs under these conditions. However, this disruption impairs the release of Rad52 from the DNA repair centers despite the lesions are repaired, leading to hypermutagenesis and sensitivity to genotoxic agents. Therefore, the MCM/RNR interactions play non-canonical roles preventing by distinct mechanisms excessive dNTP synthesis and persistent Rad52 centers.

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Project description:While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A confers cellular resistance to CHK1 inhibitors and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase which dephosphorylates the WEE1 protein and rescues WEE1 from Ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1 inhibitors. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1 inhibitor plus a WEE1 inhibitor can overcome CHK1 inhibitor resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1 inhibitor sensitivity or resistance.

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Tn-seq of hydroxyurea treated Bacillus subtilis cells

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A high-throughput screen of inactive X chromosome reactivation identifies the enhancement of DNA demethylation by 5-aza-2’-dC upon inhibition of ribonucleotide reductase-wide

Project description:Background: DNA methylation is important for maintenance of the silent state of genes on the inactive X chromosome (Xi). Here, we screened for siRNAs and chemicals that reactivate an Xi-linked reporter in the presence of 5-aza-2’-deoxycytidine (5-aza-2’-dC), an inhibitor of DNA methyltransferase 1, at a concentration that, on its own, is not sufficient for Xi-reactivation. Results: We found that inhibition of ribonucleotide reductase (RNR) induced expression of the reporter. RNR inhibition potentiated the effect of 5-aza-2’-dC by enhancing its DNA incorporation, thereby decreasing genome-wide DNA methylation levels. Since both 5-aza-2’-dC and RNR-inhibitors are used in the treatment of hematological malignancies, we treated myeloid leukemia cell lines with 5-aza-2’-dC and the RNR inhibitor hydroxyurea, and observed synergistic inhibition of cell growth and decreases in genome-wide DNA methylation. Conclusions: Taken together, our study identifies a drug combination that enhances DNA demethylation by altering nucleotide metabolism. We demonstrate that XCR assays can be used to optimize epigenetic activity of drug combinations. Reduced representation bisulfite sequencing (MspI,~40-220bp size fraction) of murine and human cells.

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