PDF(Pubmed)
Abstract：
In vitro evolution and whole genome analysis has proven to be a powerful method for studying the mechanism of action of small molecules in many haploid microbes but has generally not been applied to human cell lines in part because their diploid state complicates the identification of variants that confer drug resistance. To determine if haploid human cells could be used in MOA studies, we evolved resistance to five different anticancer drugs (doxorubicin, gemcitabine, etoposide, topotecan, and paclitaxel) using a near-haploid cell line (HAP1) and then analyzed the genomes of the drug resistant clones, developing a bioinformatic pipeline that involved filtering for high frequency alleles predicted to change protein sequence, or alleles which appeared in the same gene for multiple independent selections with the same compound. Applying the filter to sequences from 28 drug resistant clones identified a set of 21 genes which was strongly enriched for known resistance genes or known drug targets (TOP1, TOP2A, DCK, WDR33, SLCO3A1). In addition, some lines carried structural variants that encompassed additional known resistance genes (ABCB1, WWOX and RRM1). Gene expression knockdown and knockout experiments of 10 validation targets showed a high degree of specificity and accuracy in our calls and demonstrates that the same drug resistance mechanisms found in diverse clinical samples can be evolved, discovered and studied in an isogenic background.
摘要：
体外进化和全基因组分析已被证明是研究小分子在许多单倍体微生物中的作用机制的有力方法,但通常未被应用于人类细胞系,部分原因是它们的二倍体状态使赋予耐药性的变体的鉴定变得复杂。为了确定单倍体人类细胞是否可以用于MOA研究，我们进化出了对五种不同抗癌药物(阿霉素，吉西他滨,依托泊苷,托泊替康,和紫杉醇）使用近单倍体细胞系（HAP1），然后分析耐药克隆的基因组，开发一个生物信息学管道，涉及过滤预测会改变蛋白质序列的高频等位基因，或出现在同一基因中的等位基因，用于使用相同化合物进行多个独立选择。将过滤器应用于来自28个耐药克隆的序列，鉴定了一组21个基因，这些基因强烈富集了已知的耐药基因或已知的药物靶标（TOP1，TOP2A，DCK,WDR33,SLCO3A1)。此外,一些品系携带包含其他已知抗性基因(ABCB1、WWOX和RRM1)的结构变体。10个验证靶标的基因表达敲低和敲除实验在我们的调用中显示出高度的特异性和准确性，并证明在不同临床样品中发现的相同耐药机制可以进化。在同基因背景中发现和研究。
