Genome-wide genetic vulnerabilities of Flt3^ITD/+/Setbp1^IM+AMLs revealed by CRISPR knockout screening. (A) Genome-wide CRISPR-CAs9 recessive screening of 2 independent Flt3^ITD/+/Setbp1^IM+ AMLs. Cells were harvested at days 8, 12, 16, 20, and 24. Day 20 was selected as the most representative dropout screening (supplemental Figure 6A). (B) Overlap of dropout genes between the 2 Flt3^ITD/+/Setbp1^IM+ AML samples. (C) The number of depleted genes in the 2 Flt3^ITD/+/Setbp1^IM+with respect to their negative FDR (negFDR; pooled data from 2 biological replicate screenings). (D) Gene expression (RNA-seq) vs dropout P value in individual Flt3^ITD/+/Setbp1^IM+ cell lines, showing that almost all significant dropout genes (negFDR ≤0.2) are expressed at FPKM ≥0.5. (E) Overlap of dropout genes for Flt3^ITD/+/Setbp1^IM+ and Flt3^ITD/+/MLL-AF9 AMLs, and nonleukemic HPC7 hematopoietic progenitor cells, showing 420 shared pan-essential and 966 Flt3^ITD/+/Setbp1^IM+-specific dropout genes. (F) Difference in negative FDR values (ΔnegFDR) for the 966 Flt3^ITD/+/Setbp1^IM+-specific dropout genes in the HPC7 (blue) and MLL-AF9 (pink) screenings. A total of 677 genes were selected as being more specific to Flt3^ITD/+/Setbp1^IM+ AMLs, as they did not show any trend for dropping out (FDR >0.7) in either Flt3^ITD/+/MLL-AF9 or HPC7 cells. (G) GO term analysis for the 677 Flt3^ITD/+/Setbp1^IM+-specific essential genes (thresholds used: fold enrichment, >1; number of genes per set, >3; P < .05). (H) The number of druggable genes and the genes for which US Food and Drug Administration (FDA)–approved drugs are available (n = 81) according to the Drug-Gene Interaction Database (DGIdb).⁴⁵