Superenhancers shape the landscape and repair dynamics of transcription-associated DNA breaks in cancer.

Cancer is characterized by uncontrolled proliferation accompanied by oncogene hypertranscription, leading to transcription stress, a key source of DNA double-strand breaks (DSBs) that jeopardize genomic stability. Despite its importance, the landscape and consequences of transcription stress remain underexplored. Here, we used maps of DSBs identified through sBLISS (in-suspension break labeling in situ and sequencing) with transcription stress markers to delineate the transcription stress landscape in cancer. We found that transcription stress sites are shaped by the superenhancer regulatory landscape. Notably, γH2AX is enriched at transcription stress sites; however, not all DSB-enriched genes show similar γH2AX marking. Instead, genes with DSBs tied to transcription stress are distinctly marked. Genes with high DSBs marked by γH2AX exhibited substantially higher DSB turnover and repair than those with low γH2AX, and are associated with vulnerability to mutagenesis. These findings underscore superenhancer activity as a determinant of the transcription stress landscape in cancer, posing a threat to the genomic stability of oncogenes.