How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.