LMO1 enhancer mutation analyses for transcription activity in vitro and leukemia progression in vivo. (A) Jurkat cells were transfected with a reporter gene construct with WT sequence or LMO1 enhancer mutation, and transcription activation was quantified by luciferase activity relative to Renilla. LMO1 mutant enhancer activity was significantly diminished upon downregulation of MYB by short interfering RNA (siRNA). (B) In HEK293T cells, LMO1 enhancer mutation drove luciferase transcription only when MYB was ectopically expressed. (C) LMO1 overexpression was abrogated upon conversion of the LMO1 mutant allele to the WT allele in Jurkat cells, using CRISPR/Cas9 genomic engineering. (D) Engineered Jurkat cells with WT genotype at the LMO1 enhancer locus also showed a significantly lower rate of proliferation compared with parental Jurkat cells. (E) Jurkat cells were transduced with lentiviral vectors encoding constitutive Cas9 and isgLMO1. Parental Jurkat or CRISPR/Cas9-engineered cells were injected into sublethally irradiated NOD.Cg-Prkdc^scidII2rg^tm1Wjl/SzJ recipient mice. Mice bearing genome-edited Jurkat cells were split into 2 groups: with vs without doxycycline (Dox) treatment (ie, with vs without LMO1 enhancer deletion). (F) Leukemia progress was monitored weekly by complete blood count and flow cytometry quantification of human CD45 cells in peripheral blood. Median survival for mice bearing parental cells or isgLMO1 cells without doxycycline treatment was 29.5 and 32.5 days, respectively, whereas doxycycline-treated mice bearing isgLMO1 cells remained alive at the end of this experiment. Statistical significance of the differences was estimated by using 2-sided Mann-Whitney-Wilcoxon test (A-C, E), 2-way analysis of variance (D), or log-rank test (F); *P < .05, **P < .01, and ***P < .001. NS, not significant.