![ERG is a direct transcriptional target of EVI1 in murine and human AML. (A) Genome-wide CRISPR/Cas9-based loss-of-function screens in murine RUNX1/EVI1 vs MLL/AF9-driven leukemia cells. Gray dots illustrate all genes. Dark blue dots represent conserved transcriptional targets of EVI1. Axis shows FDR-adjusted normalized log2 FC. Dashed box represents genes with an FDR-adjusted normalized log2 FC < −0.5 in murine RUNX1/EVI1-driven and > −0.5 in murine MLL/AF9-driven AML. (B) Genome-wide CRISPR/Cas9-based loss-of-function screens in murine RUNX1/EVI1-driven vs human HNT-34 AML cells. Gray dots illustrate all genes. Dark blue dots represent conserved transcriptional targets of EVI1. Axis shows FDR-adjusted normalized log2 FC. Dashed box represents genes with an FDR-adjusted normalized log2 FC < −0.5 in murine RUNX1/EVI1-driven AML and < −0.5 in human HNT-34 cells. (C) Comparative analysis of loss-of-function–mediated gene effects in human and murine EVI1–dependent AML compared with all other AML cell lines. Gray dots represent all genes. Dark blue dots represent conserved transcriptional targets of EVI1. X-axis depicts the average differential gene effect between the 2 groups. Y-axis depicts the log-transformed Bayesian factor. (D) Expression of ERG in human HNT-34 cells 5 days upon shRNA-mediated EVI1 knockdown in vitro and in murine ex vivo–derived RUNX1/EVI1-driven leukemia cells extracted from the BM of mice upon 3 days of Dox treatment (mean ± standard deviation [SD]; n = 3). (E) Flow cytometric analysis of intracellular RUNX1/EVI1 and ERG levels in murine leukemia cells 5 days upon RUNX1/EVI1 knockdown compared with nontargeting and isotype control. (F) Quantification of the mean fluorescence intensity of RUNX1/EVI1 and ERG levels as determined by flow cytometry in murine leukemia cells 5 days upon RUNX1/EVI1 knockdown, compared with knockdown of Myc and nontargeting shRen.713 (mean ± SD; n = 3). (G) EVI1 occupancy at the ERG locus. H3K27ac ChIP-seq in HNT-34 and MOLM-1 (gray). EVI1 ChIP-seq in HNT-34, MOLM-1 and primary patient-derived AML cells (EVI1-R, blue). ATAC-seq in patient-derived AML cells (red). The +85 stem cell enhancer region43 is indicated. ns, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/141/5/10.1182_blood.2022016592/6/blood_bld-2022-016592-gr4.jpeg?Expires=1718244334&Signature=iIjNX8SGrLwQAeo9HOC1qRj5OEGexxKKzysTqDWiyM-kbZ4am3-K9afwcGMapl5IxwbvtwHbQ3EdCDccuYIHLcpwuhqCaFPIsjuvRJwzUnT2vdPXnUZB6~qszgsuAA5MA2GytdVgNR-r0TApGD-kQBRJqWRxNXPW0lpWtbgH~tCMDlEK~RsLdOIcBOMKZLtZMuARgIA3s3-V3aBFo3ac5FatLjdXa0tcQrlNcL3W7pUZdkQlm4Wf3v8Y3Qe21uw1h-ZhKsaEswXzI3029~XMWtwLDNq89L-ZyG0dQux35uvVlJ0lnVyQGLdusnZwnrana51NfRJD1jujrNdSVz-4Ww__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ERG is a direct transcriptional target of EVI1 in murine and human AML. (A) Genome-wide CRISPR/Cas9-based loss-of-function screens in murine RUNX1/EVI1 vs MLL/AF9-driven leukemia cells. Gray dots illustrate all genes. Dark blue dots represent conserved transcriptional targets of EVI1. Axis shows FDR-adjusted normalized log2 FC. Dashed box represents genes with an FDR-adjusted normalized log2 FC < −0.5 in murine RUNX1/EVI1-driven and > −0.5 in murine MLL/AF9-driven AML. (B) Genome-wide CRISPR/Cas9-based loss-of-function screens in murine RUNX1/EVI1-driven vs human HNT-34 AML cells. Gray dots illustrate all genes. Dark blue dots represent conserved transcriptional targets of EVI1. Axis shows FDR-adjusted normalized log2 FC. Dashed box represents genes with an FDR-adjusted normalized log2 FC < −0.5 in murine RUNX1/EVI1-driven AML and < −0.5 in human HNT-34 cells. (C) Comparative analysis of loss-of-function–mediated gene effects in human and murine EVI1–dependent AML compared with all other AML cell lines. Gray dots represent all genes. Dark blue dots represent conserved transcriptional targets of EVI1. X-axis depicts the average differential gene effect between the 2 groups. Y-axis depicts the log-transformed Bayesian factor. (D) Expression of ERG in human HNT-34 cells 5 days upon shRNA-mediated EVI1 knockdown in vitro and in murine ex vivo–derived RUNX1/EVI1-driven leukemia cells extracted from the BM of mice upon 3 days of Dox treatment (mean ± standard deviation [SD]; n = 3). (E) Flow cytometric analysis of intracellular RUNX1/EVI1 and ERG levels in murine leukemia cells 5 days upon RUNX1/EVI1 knockdown compared with nontargeting and isotype control. (F) Quantification of the mean fluorescence intensity of RUNX1/EVI1 and ERG levels as determined by flow cytometry in murine leukemia cells 5 days upon RUNX1/EVI1 knockdown, compared with knockdown of Myc and nontargeting shRen.713 (mean ± SD; n = 3). (G) EVI1 occupancy at the ERG locus. H3K27ac ChIP-seq in HNT-34 and MOLM-1 (gray). EVI1 ChIP-seq in HNT-34, MOLM-1 and primary patient-derived AML cells (EVI1-R, blue). ATAC-seq in patient-derived AML cells (red). The +85 stem cell enhancer region43 is indicated. ns, not significant.
