Validation of select dropout genes in vitro and in vivo. (A) CRISPR-gRNA validation of 26 dropout and 7 enriched genes belonging to different genetic categories, using competitive coculture, counting the percentage of BFP⁺ (transduced) vs percentage of BFP⁻ (nontransduced) cells at each time point. (B) Outline of in vivo validation approach using CRISPR-gRNA in a stable luciferase-expressing Flt3^ITD/+/Setbp1^IM+ AML cell line and transplantation into NSG mice. (C) Bioluminescence images of representative transplant-recipient mice from each single gRNA cohort. Flt3^ITD/+/Setbp1^IM+AML cells transduced with gRNAs against the dropout genes Hoxa9, Setbp1, Erg, and Hmgrc were associated with a trend toward reduced luminescence at 6 weeks and with a significantly prolonged survival compared with cells transduced with an empty vector. By contrast, cells transduced with a gRNA targeting the enriched gene Bcor, had extensive engraftment by 6 weeks and exhibited a trend toward reduced survival. Median survivals were Hoxa9, 102 days; Setbp1, 79 days; Hmgcr, 69 days; Erg, 130 days; and empty control, 57 days, Bcor, 52 days. P values: Student t test for luminescence and Mantel-Cox for survival. (D) Dose-response curves for iBET-151 and KDM1A inhibitors ORY-1001 and GSK-LSD1. Parental Flt3^ITD/+/Setbp1^IM+ AMLs (solid lines) and their Cas9-expressing clones (dotted lines) show higher sensitivity to iBET-151 compared with 2 independent Flt3^ITD/+/Npm1^cA/+ AMLs (blue/purple). The differences in responses to ORY-1001 and GSK-LSD1 treatment were more marked, with Flt3^ITD/+/Setbp1^IM+ AMLs showing significant sensitivity, whereas Flt3^ITD/+/Npm1^cA/+ AMLs showed no notable response to these compounds over the concentration range tested. ND, not determined.