R-Loop-Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin

Area of Science:

• Molecular Biology
• Cancer Biology
• Epigenetics

Background:

• Histone deacetylase inhibitors (HDACi) are approved for hematological cancers but show limited efficacy in solid tumors.
• Understanding the precise mechanisms of action (MOA) of HDACi is crucial for improving their therapeutic potential.
• Beyond gene expression, HDACi exhibit diverse cellular activities influenced by cell context.

Purpose of the Study:

• To elucidate the dominant DNA damage signature induced by HDACi in solid tumors.
• To investigate the molecular mechanisms by which histone hyperacetylation leads to DNA damage.
• To explore the potential of targeting DNA repair pathways in combination with HDACi therapy.

Main Methods:

• Utilized the NCI-60 cell line database to identify DNA damage as a key signature.
• Employed single-cell electrophoresis to detect single-stranded DNA (ssDNA) breaks.
• Investigated the role of transcription-coupled base excision repair (BER) in resolving DNA damage.
• Assessed the impact of inhibiting BER proteins, such as PARP, on DNA double-strand breaks (dsDNA).

Main Results:

• Established DNA damage as the predominant cellular response to HDACi using a clinically relevant exposure duration.
• Identified the accumulation of DNA-RNA hybrids (R-loops) as a direct consequence of romidepsin-induced histone hyperacetylation.
• Demonstrated that unrepaired ssDNA breaks can evolve into lethal dsDNA breaks when the BER pathway is overwhelmed.
• Showed that inhibiting BER proteins, like PARP, exacerbates dsDNA breaks in the presence of HDACi.

Conclusions:

• Romidepsin-induced histone hyperacetylation leads to the accumulation of R-loops, a novel mechanism of DNA damage.
• Transcription-coupled BER plays a role in repairing ssDNA breaks, but its capacity can be exceeded, leading to dsDNA breaks.
• Targeting BER pathways, such as PARP inhibition, in conjunction with HDACi may enhance therapeutic efficacy against solid tumors.
• These findings provide critical insights for designing improved combination therapies involving HDAC inhibitors for solid tumor treatment.