Robust methods are needed for determining the mechanism of action and mechanism of resistance for small molecules that are active against human cells. In microbes, in vitro evolution and whole genome analysis (IVIEWGA) is the preferred method for target deconvolution and for revealing resistance mechanisms. To investigate the method’s applicability to human cells we evolved resistance to five different anticancer drugs (doxorubicin, gemcitabine, etoposide, topotecan, and paclitaxel) using a near-haploid cell line (HAP-1). Resultant clones (N=28) were compared to their isogenic parents via whole genome and whole exome sequencing (WES). High frequency alleles predicted to change protein sequence, or alleles which appeared in the same gene for multiple independent selections with the same compound were identified in only 21 genes: The set included clinically-relevant resistance genes or known drug targets (TOP1, TOP2A, DCK, WDR33, SLCO3A1), as well as new genes (SLC13A4). In addition, some lines carried structural variants that encompassed additional known resistance genes (ABCB1, WWOX and RRM1). Gene expression knockdown and knockout experiments (via shRNA and CRISPR- Cαs 9 respectively) 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, identified and studied in an isogenic background. Our data show that in vitro evolution and whole genome analysis is a promising method for target identification as well as for identifying resistance mechanisms. ### Competing Interest Statement The authors have declared no competing interest. * AF : allele frequency; CNV : Copy Number Variation; NGS : Next Generation Sequencing, WES : Whole Exome Sequencing; WGS : Whole Genome Sequencing; CML : Chronic Myelogenous Leukemia; IVIEWGA : In Vitro Evolution and Whole Genome Analysis; SNV : Single Nucleotide Variant; CNV : Copy Number Variation; TF : transcription factor; DOX : Doxorubicin; GEM : Gemcitabine; ETP : Etoposide; PTX : Paclitaxel; TPT : Topotecan; AML : Acute Myeloid Leukemia; TKIs : Tyrosine Kinase Inhibitors; MDR : Multi-Drug Resistance; gDNA : genomic DNA.
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