Persistent TOP1 cleavage complexes in drug-tolerant cells drive adaptive resistance to EGFR-targeted therapies in lung cancer

Resistance to targeted cancer therapies often arises from drug-tolerant cells (DTCs), which survive treatment by entering a non-proliferative state. Over time, DTCs can acquire mutations that contribute to cell reproliferation, but how non-proliferating DTCs accumulate such mutations remains unclear. Here, we show that EGFR inhibition in EGFR-mutated lung cancer transiently downregulates tyrosyl-DNA phosphodiesterase 1 (TDP1), a repair enzyme that resolves abortive topoisomerase I cleavage complexes (TOP1ccs). In DTCs, elevated reactive oxygen species promote TOP1cc trapping, while TDP1 downregulation impairs their repair, driving TOP1cc accumulation, resistance mutation acquisition, and cell reproliferation. We further find that TDP1 expression is absent in approximately 25% of EGFR-mutated lung cancers. In TDP1-deficient cells, combining EGFR inhibition with a sublethal concentration of topotecan, which further increases TOP1ccs, abolishes cell reproliferation. Together, these findings establish persistent TOP1cc accumulation as a driver of therapy-induced mutagenesis linking drug tolerance to adaptive resistance, and reveal TDP1 loss as a targetable vulnerability in EGFR-mutated lung cancers. TeaserA drug-tolerant state that fuels mutagenesis and resistance also creates a transient vulnerability.