Homologous recombination repair creates mutations in the non-coding genome that alter Topoisomerase-1 cleavage sites & orchestrates irinotecan resistance

Abstract 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, selection of drug-resistant mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells, and thus cannot result from selection of pre-existent mutations. Accordingly, adaptation must involve 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. Resistance mechanism involved gradual accumulation of recurrent mutations in non-coding regions of DNA at Top1-cleavage sites. Surprisingly, cancer cells had higher number of such sites than reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repair 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 generation of DNA breaks upon subsequent exposures, thus gradually increasing the drug resistance. Together, large target size for mutations and their Top1-guided generation lead to their gradual and rapid accumulation, synergistically accelerating development of resistance. Abstract Figure