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

Abstract

AbstractDevelopment of resistance to chemotherapy is a leading cause of treatment failure. Drug-resistance expression. It is commonly understood that such mutations happen randomly prior to treatment and are positively selected during the treatment. We realized that selection of drug-resistant cancer cell mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells, and thus is unlikely to result from selection of pre-existent mutations. Accordingly, adaptation must involve generation of mutations de-novo in the process of drug treatment. Here we explored both the mechanism of resistance and the origin of resistant mutations in response to a widely used Top1 inhibitor irinotecan, which triggers DNA breaks, resulting in cytotoxicity. Resistance mechanism was based on gradual accumulation of recurrent mutations in non-coding regions of DNA at sequence-specific Top1 cleavage sites. Surprisingly, cancer cells had much 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 cleavage sites following initial drug exposures gradually reverted cleavage-sensitive cancer-related sequences back to cleavage-resistant “normal” genome sequences. These genetic changes reduced generation of double strand DNA breaks upon subsequent exposures, thus gradually increasing the drug resistance. Together, large target size for resistance mutations and their generation specifically at Top1 cleavage spots lead to gradual and rapid accumulation of such mutations, synergistically accelerating development of resistance.Graphical abstract