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

The selection of drug-resistant mammalian cell mutants requires multiple drug exposures. Since cells in starting population could be genetically identical, selection of pre-existent mutations is unlikely. Therefore, adaptation must involve the generation of drug-resistant mutations de-novo. Understanding how adaptive mutations are generated and protect cells is important for our knowledge of cancer biology and evolution. Here, we studied the adaptation of cancer cells to topoisomerase (Top1) inhibitor irinotecan, which triggers DNA breaks, resulting in cytotoxicity. The resistance mechanism was based on the gradual accumulation of recurrent mutations in non-coding DNA at sequence-specific Top1 cleavage sites. Repair of DSBs at these sites following initial irinotecan exposures created mutant sequences that were resistant to further Top1 cleavage. Therefore, by virtue of creating DNA breaks Top1 increases the rate of highly protective mutations specifically at such spots, thus explaining a puzzling need for dose escalation in resistance development.

耐药性哺乳动物细胞突变株的筛选需要经历多次药物暴露。由于起始细胞群内的细胞可具备完全一致的基因型，筛选预先存在的突变几乎不可能发生。因此，细胞的适应性演化必然依赖于新发（de novo）耐药突变的产生。解析适应性突变的产生机制及其对细胞的保护作用，对于我们理解癌症生物学与演化过程具有重要意义。
本研究针对癌细胞对拓扑异构酶1（Top1）抑制剂伊立替康的适应性响应展开探究：伊立替康可诱导DNA断裂，进而引发细胞毒性。该耐药机制的核心是，在序列特异性Top1切割位点处的非编码DNA区域内，复发性突变会逐步积累。在首次暴露于伊立替康后，这些位点的DNA双链断裂（double-strand breaks, DSBs）的修复过程会产生对后续Top1切割具有抗性的突变序列。因此，Top1通过诱导DNA断裂，可特异性提升此类位点上高保护性突变的发生频率，这也解释了耐药性发展过程中令人困惑的剂量递增需求。