Evolution of Resistance to Irinotecan in Cancer Cells Involves Generation of Topoisomerase-Guided Mutations in Non-Coding Genome That Reduce the Chances of DNA Breaks

Resistance to chemotherapy is a leading cause of treatment failure. Drug resistance mechanisms involve mutations in specific proteins or changes in their expression levels. It is commonly understood that resistance mutations happen randomly prior to treatment and are selected during the treatment. However, the selection of drug-resistant mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells and thus cannot result from the selection of pre-existent mutations. Accordingly, adaptation must involve the generation of mutations de novo upon drug treatment. Here we explored the origin of resistance mutations to a widely used Top1 inhibitor, irinotecan, which triggers DNA breaks, causing cytotoxicity. The resistance mechanism involved the gradual accumulation of recurrent mutations in non-coding regions of DNA at Top1-cleavage sites. Surprisingly, cancer cells had a higher number of such sites than the reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repairs of DNA double-strand breaks at these sites following initial drug exposures gradually reverted cleavage-sensitive cancer sequences back to cleavage-resistant normal sequences. These mutations reduced the generation of DNA breaks upon subsequent exposures, thus gradually increasing drug resistance. Together, large target sizes for mutations and their Top1-guided generation lead to their gradual and rapid accumulation, synergistically accelerating the development of resistance.

Automated summary

Resistance to chemotherapy is commonly caused by mutations or changes in protein expression. The selection of drug-resistant mutants can occur randomly prior to treatment or through the generation of mutations during treatment. Here, we studied the origin of resistance mutations to the Top1 inhibitor irinotecan and found that the mutations accumulated in non-coding regions of DNA at Top1-cleavage sites. Cancer cells had a higher number of these sites compared to the reference genome, contributing to their increased sensitivity to irinotecan. These mutations gradually reduced the generation of DNA breaks and increased drug resistance.