An old drug gets a new look: DNA damaging agent aclarubicin found to disrupt chromatin and stimulate RNA Pol II elongation

From the Henikoff Lab, Basic Sciences Division and Cancer Basic Biology Program of the Cancer Consortium

Originally isolated from soil microbes, “anthracyclines rapidly made their way to the clinic and are still one of the most effective compounds we have today,” explained Dr. Matthew Wooten, a postdoctoral researcher in the Henikoff Lab. Anthracyclines are a class of drugs which include the common chemotherapy drug doxorubicin as well as its lesser known cousin aclarubicin, which will become “the hero of this story,” as Wooten described. Most anthracyclines poison topoisomerase, an enzymatic detangler of DNA knots, which is lethal to the cell when these tangles can’t be repaired due to the amount of DNA damage caused. Or so scientists thought… In recent years, studies, including ones from the Henikoff Lab, have begun to challenge this model and demonstrated that there is more to the picture than just DNA damage. It turns out that chromatin disruption may be a bigger culprit of cell death for certain types of anthracyclines, including aclarubicin. However, it has remained unclear exactly how extensive anthracycline-induced chromatin damage is, and how this type of chromatin disruption might affect downstream gene expression. In a recent study published in Science Advances, Wooten and researchers from the Henikoff group investigated how different anthracyclines affect the chromatin landscape.

Working with Drosophila cells, where transcriptional regulation has been extensively characterized, Wooten and colleagues treated cells with doxorubicin or aclarubicin, in addition to actinomycin D, a non-anthracycline intercalating agent known to arrest RNA Polymerase II (Pol II) elongation. The authors first sought to understand how these drugs affected RNA Pol II dynamics and assessed genomic distribution of both paused and elongating Pol II using the Henikoff lab’s bread and butter technology- CUT&Tag. While the amount and distribution of paused Pol II were similar across all three treatments, aclarubicin-treated samples gained large amounts of elongating Pol II around active promoters, with actinomycin D, but not doxorubicin, also gaining modest amounts of elongating Pol II. Wooten admitted this result was a bit of a letdown and might support a model where aclarubicin and actinomycin D work in a similar manner. However, thanks to a useful reviewer comment, when Wooten looked at levels of total Pol II, he found aclarubicin-treated samples showed increased total Pol II levels. In contrast, actinomycin D-treated samples showed a decrease, supporting the hypothesis that the two drugs do indeed work by different mechanisms. With data analysis help from research technician Brittany Takushi, Wooten investigated whether this aclarubicin-induced increase in Pol II affected chromatin accessibility and G-quadruplex formation- both of which are known to be associated with changes in Pol II occupancy. To assay chromatin accessibility changes, the researchers used another Henikoff lab-developed tool, CUTAC (Cleavage Under Targeted Accessible Chromatin), which alters CUT&Tag salt conditions to identify accessible DNA regions adjacent to RNA Pol II. Comparing G-quadruplexes (identified with CUT&Tag) and accessible chromatin regions, they found that treatment with aclarubicin uniquely affected certain clusters of genes. These included increases in both accessibility and G-quadruplex formation at some genes, and decreases in both of these chromatin attributes at others. These corresponding relationships between accessibility and G-quadruplex formation were not seen in cells treated with doxorubicin or actinomycin D.

model for aclarubicin-induced chromatin damage
Model for aclarubicin-induced chromatin damage. Image provided by Dr. Matthew Wooten

Genome connoisseurs like Wooten typically prefer scrolling through the genome over scrolling through social media feeds, although he admits that this can be an equally fruitless task. However, in one particular wandering through the genome trying to figure out why accessibility of certain genes seemed to be more affected by aclarubicin than others, he noticed something intriguing. Interestingly, it didn’t seem to be that the most accessible regions were necessarily affected. Rather, he found that closely spaced gene regions, specifically divergent gene promoters, or neighboring genes that are oriented in opposite directions, were particularly victimized by aclarubicin-induced chromatin changes. Wooten explained that when genes are going in opposite directions, it creates a great amount of torsion, or negative super coiling, leaving a literal gaping opportunity for aclarubicin to slide right in.

To gain further insight in how aclarubicin might affect certain regions of the genome with particular structural attributes, the research team analyzed Dodeca-satellite tandem repeats found in pericentromeric chromatin as well as the histone locus cluster- a region of the genome with closely spaced divergent promoters that express histone genes. While the Dodeca-satellite repeats, known for noncanonical chromatin structures, were particularly sensitive to aclarubicin-induced G-quadruplex formation, the histone locus was not. Instead, the histone locus showed increased chromatin accessibility and elongating Pol II, demonstrating that aclarubicin can induce distinct responses at certain gene regions and that the strongest impact of aclarubicin treatment is on elongating Pol II. Furthermore, since high levels of free histones can induce cell death, this may contribute to the toxicity of aclarubicin.

With our growing knowledge of the epigenome and its importance for gene regulation in cancers, there is a push to develop epigenetic therapeutics. For drugs that have been around for a while like aclarubicin, Wooten remarked on the irony that “we’ve potentially have been using epigenetic drugs this whole time, we just didn’t know it.” Aclarubicin has been around since the 1980s, yet for unclear reasons isn’t widely used in the US. Whereas in Asia, it’s commonly used to treat elderly cancer patients and patients with heart problems that can’t tolerate more toxic chemotherapies. In line with this, doxorubicin is one of the front-line treatments for pediatric cancers, but can have long-term adverse effects on the heart. For this reason, Cancer Consortium members Drs. Beth Lawlor and Jay Sarthy, both Principal Investigators at Seattle Children’s, are interested in aclarubicin for a less toxic, but still effective, treatment for pediatric cancers. Together with Lawlor and Sarthy, Wooten is continuing his research on aclarubicin to understand exactly how this chromatin-disrupting drug causes cell death, with the hope of ultimately understanding whether this drug might be a good candidate for these childhood cancers.

This work was supported by the Howard Hughes Medical Institute and the University of Washington Genome Training Grant.

UW/Fred Hutch/Seattle Children’s Cancer Consortium member Steven Henikoff contributed to this work.

Wooten M, Takushi B, Ahmad K, Henikoff S. Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters. Sci Adv. 2023 Jun 16;9(24):eadg3257. doi: 10.1126/sciadv.adg3257.