Genome-scale knockout screening reveals genes that meditate crizotinib resistance in anaplastic large cell lymphoma (ALCL). (A) Experimental design of the crizotinib resistance screen in ALK⁺ ALCL cell lines using the GecKO v2 library. (B) Genes ranked by MAGeCK Robust Rank Aggregation enrichment score in crizotinib over DMSO-treated cells, highlighting the top 10 of candidate genes acquired following specific filtering criteria based on the ranking list (see "Materials and methods"). ***FDR ≤0.01, **FDR ≤0.1, *FDR ≤ 0.2. (C) Deletion of PTPN1 or PTPN2 in ALK⁺ ALCL cell lines. TS and SU-DHL-1 cells were transduced with 2 different sgRNAs targeting either PTPN1 or PTPN2. Cells were treated with 80 nM or 300 nM crizotinib to evaluate changes in PTPN1 or PTPN2 expression. Western blotting was performed on cell lysates probed with the indicated antibodies. β-actin or β-tubulin were used as loading controls. One representative experiment of 4 is shown. (D) Cell viability assay was performed on TS and SU-DHL-1 WT or transduced cells with either PTPN1 or PTPN2 targeting sgRNAs undergoing crizotinib treatment. Data are represented as mean ± standard deviation (SD) of technical triplicates; **P < .01, ***P < .001, ****P < .0001, 2-way ANOVA followed by Dunnett's multiple comparisons test. (E) Growth curves of WT, PTPN1 KO, or PTPN2 KO ALK⁺ cells (TS and SU-DHL-1) treated with 80 nM crizotinib. Data are represented as means ± SD of technical triplicates; ****P < .0001, 2-way ANOVA followed by Tukey's multiple comparisons test. (F) PTPN1 KO or PTPN2 KO TS cells were subcutaneously injected into NSG mice. The mice were treated with crizotinib (75 mg/kg daily) by oral gavage. Data are represented as means ± SD of 4-6 mice in the control groups and 8 mice in the crizotinib-treated groups; ****P < .0001, 2-way ANOVA followed by Tukey's multiple comparisons test.