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A high-throughput screening identifies MCM chromatin loading inhibitors targeting cells with increased replication origins.

ABSTRACT: Replication origin assembly is a pivotal step in chromosomal DNA replication. In this process, the ORC complex binds DNA and, together with the CDC6 and CDT1, promotes the loading of the MCM helicase. Chemicals targeting origin assembly might be useful to sensitize highly proliferative cancer cells. However, identifying such compounds is challenging due to the multistage nature of this process. Here, using Xenopus laevis egg extract we set up a high-throughput screening to isolate MCM chromatin loading inhibitors, which led to the identification of NSC-95397 as a powerful inhibitor of replication origin assembly that targets CDC6 protein and promotes its degradation. Using systems developed to test selective drug-induced lethality we show that NSC-95397 triggers cell death both in human cells and Xenopus embryos that have higher proliferative ability. These findings demonstrate the effectiveness of molecules disrupting DNA replication processes in targeting hyperproliferating cells, highlighting their potential as anti-cancer molecules.

SUBMITTER: Falbo L

PROVIDER: S-EPMC11342271 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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A high-throughput screening identifies MCM chromatin loading inhibitors targeting cells with increased replication origins.

Falbo Lucia L Técher Hervé H Sannino Vincenzo V Robusto Michela M Fagà Giovanni G Pezzimenti Federica F Romeo Francesco F Colombo Luca Gabriele LG Vultaggio Stefania S Fancelli Daniele D Monzani Silvia S Cecatiello Valentina V Pasqualato Sebastiano S Varasi Mario M Mercurio Ciro C Costanzo Vincenzo V

iScience 20240722 8

Replication origin assembly is a pivotal step in chromosomal DNA replication. In this process, the ORC complex binds DNA and, together with the CDC6 and CDT1, promotes the loading of the MCM helicase. Chemicals targeting origin assembly might be useful to sensitize highly proliferative cancer cells. However, identifying such compounds is challenging due to the multistage nature of this process. Here, using <i>Xenopus laevis</i> egg extract we set up a high-throughput screening to isolate MCM chr ...[more]

PMID: 39184446

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Project description:BACKGROUND: Previous studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521. RESULTS: Screening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5? strains show a G1 delay after CG-1521 treatment, however the gcn5? strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5? strain. CONCLUSION: Genome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.

Dataset Information

A high-throughput screening identifies MCM chromatin loading inhibitors targeting cells with increased replication origins.

Publications

A high-throughput screening identifies MCM chromatin loading inhibitors targeting cells with increased replication origins.

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