Role Of Chromatin Damage In Mediating The Anticancer Cytotoxicity Of Dna-Binding Small Molecule Drugs

DNA-targeting small molecules are chemicals with different mechanisms of action combined by a common outcome - DNA damage. Since cancer cells are very vulnerable to DNA damage, many DNA-targeting small molecules are used for cancer treatment. The DNA-damaging effect of small molecules depends on chromatin organization, however, the exact mechanism by which small molecules affect the chromatin, as well as the role of chromatin alterations in their biological activity is poorly understood.

Previously we identified a small molecule, curaxin CBL0137, which has broad anti-cancer activity, and binds DNA without detectable DNA damage by interfering with histone/DNA interactions followed by nucleosome disassembly in vitro and in cells, the phenomenon which we named “chromatin damage”. This CBL0137-induced disassembly of the nucleosome is recognized by the histone chaperone FACT (FAcilitates Chromatin Transcription), whose normal function is to control nucleosome stability. We named this massive binding of FACT to different components of disassembled chromatin in curaxin-treated cells “c-trapping”.

This study was based on the hypothesis that curaxins may not be unique in their ability to cause chromatin damage and c-trapping. We selected a set of DNA-targeting agents, representing different modes of DNA binding and inhibition of topoisomerases: inhibitors of topoisomerases cleavage activity, inhibitors of topoisomerases II re-ligation, compounds that do not bind DNA directly while form a cleavable complex with DNA and topoisomerases I or II, as well as topoisomerases II catalytic inhibitors that do not bind DNA. We identified cytotoxicity of compounds in several human cell lines and used western blotting to detect c-trapping, further confirmed by fluorescent imaging of live cells and immunofluorescent staining. Using recombinant mononucleosomes and micrococcal nuclease assays we analyzed nucleosome stability and chromatin destabilization by the tested drugs. DNA – damaging activity of tested compounds was also evaluated.

We found that only DNA direct binding compounds are able to induce c-trapping, which in turn correlates with nucleosome destabilization of tested compounds and their cytotoxicity. We demonstrated that damage of chromatin may occur independently of DNA damage and is reliant on the compound’s mechanism of DNA binding. We showed that FACT is sensitive to a different stages of nucleosomes disassembly initiated by DNA-binding small molecules, including the linker histone removal followed by core histone`s eviction, and negative supercoiling accumulation. We suggest that chromatin disruption induced by small molecules plays a more important role in the toxicity of DNA binding molecules than DNA-damaging by itself.