(a–f) Loss-of-function genetic screens were performed and displayed as described in Figure 1a. Cells were treated with anticancer drugs at the concentration specified in each panel. Green bubbles represent genes known to be involved in the mechanism of action of the drug tested and the diameter of the bubble is proportional to the number of unique insertion sites in the pooled resistant clones (doxorubicin, TOP2A: N = 4; etoposide, TOP2A: N = 5; topotecan, TOP1: N = 10; bortezomib, PSMC6: N = 8; oxaliplatin, SLC43A2: N = 5; gemcitabine, DCK: N = 161). Genes with a total of less than 50 sequencing reads were not displayed in the bubble plots. (a) The screen with doxorubicin uncovered TOP2A, which encodes topoisomerase IIA, the direct target of doxorubicin (Tewey et al., 1984). (b) The screen with etoposide uncovered TOP2A, consistent with its known mechanism of action (Montecucco and Biamonti, 2007). (c) The screen with topotecan identified its known direct target (Pommier, 2006), type I topoisomerase (TOP1), as well as ABCG2. It is known that overexpression, not inactivation, of this transporter causes resistance to topotecan (Mao and Unadkat, 2015). In the pooled resistant clones, retroviral integration sites clustered to the beginning of the ABCG2 transcript and most were actually found upstream of the transcription start site (TSS). A topotecan-resistant clone (ABCG2GT) with a retroviral site 400 bp upstream of the TSS of ABCG2 was isolated and the expression level of ABCG2 was determined by RT-qPCR (Materials and methods). ABCG2GT displayed a △△CT of 6.2 ± 0.3 compared to KBM7 indicating an increase in ABCG2 mRNA levels of up to 70-fold compared to KBM7 (mean values of three independent experiments ± standard deviation). (d) The screen with bortezomib, a proteasome inhibitor (Adams, 2004), identified proteasome subunits (PSMC6, PSMD12, PSMC5, PSMD7, PSMD2). (e) The screen with oxaliplatin, a DNA crosslinker, identified SLC43A2, a solute carrier that transports neutral amino acids into cells (Bodoy et al., 2013), suggesting that SLC43A2 facilitates the transport of oxaliplatin into cells consistent with the known role of solute carriers in the transport of anticancer drugs (Li and Shu, 2014). (f) The screen with gemcitabine, a deoxycytidine analog, identified DCK and CDADC1. Deficiency of DCK is associated with gemcitabine resistance (Bergman et al., 2002) and CDADC1 is a cytidine and dCMP deaminase known to be involved in resistance to deoxycytidine analogs (Cai et al., 2008) (Figure 1—figure supplement 2—source data 1).