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, which survive treatment by entering a nonproliferative state. Over time, these cells can acquire mutations that contribute to cell reproliferation, but how nonproliferating drug-tolerant cells accumulate these mutations remains unclear. Here, we show that EGFR inhibition in EGFR-mutated lung cancer transiently down-regulates tyrosyl-DNA phosphodiesterase 1 (TDP1), a repair enzyme that resolves abortive topoisomerase I cleavage complexes (TOP1ccs). In drug-tolerant cells, elevated reactive oxygen species promote TOP1cc trapping, while TDP1 down-regulation impairs their repair, driving TOP1cc accumulation, resistance mutation acquisition, and cell reproliferation. We further find that TDP1 expression is absent in ∼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.