Cyclin dependent kinase 9 inhibitor induces transcription-replication conflicts and DNA damage accumulation in breast cancer

TL;DR

This study shows that a CDK9 inhibitor causes DNA damage in breast cancer cells by creating conflicts between transcription and replication, and identifies a protein that helps resolve these conflicts.

Contribution

The study reveals a novel mechanism of CDK9 inhibition-induced DNA damage and identifies DDX25 as a key mediator in resolving transcription-replication conflicts.

Findings

AZD4573 induces T-R conflicts and DNA damage during S-phase, leading to apoptosis in breast cancer cells.

DDX25 helicase resolves T-R conflicts and reduces sensitivity to AZD4573.

CDK9 inhibition causes replication stress and DNA strand breaks in breast cancer cells.

Abstract

Cyclin-dependent kinase 9 (CDK9) is a crucial regulator of transcriptional progression of RNA polymerase-II (RNAP2). RNA polymerases trapped in DNA can be a source of transcription-replication conflict (T-R conflict), which is a common source of replication stress. AZD4573, a highly selective CDK9 inhibitor, has been shown to induce apoptosis in leukemia cell lines, while its anti-tumor potential in breast cancer has yet to be elucidated.

To evaluate the cytotoxicity of AZD4573 in vitro, MTT assays were performed. The expression of signal transduction molecules was determined using Western blotting, immunoprecipitation, and immunofluorescence. Apoptotic cell death was verified by the annexin-V assay. DNA strand breaks and repair efficacy were evaluated through the alkaline comet assay. The siRNA knock-down system was used to confirm the action mechanism.

AZD4573 induced T-R conflicts during S-phase, increasing replication stress and DNA strand breaks, resulting in apoptosis by induction of caspase-3. Furthermore, we identified Dead-box 25 (DDX25) helicase as a key mediator in resolving the T-R conflicts. Nuclear translocation of DDX25 correlated with reduced sensitivity to AZD4573 by the resolution of T-R conflicts.

Inhibition of CDK9 by AZD4573 induces the accumulation of DNA damage through T-R conflicts. DDX25 helicases were identified as a key mediator in resolving T-R conflicts and the reduced sensitivity to AZD4573.

The online version contains supplementary material available at 10.1186/s12935-025-03897-6.

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