Loss of PRC1.1 or PRC2.2 renders menin-MLL inhibitor resistance in KMT2A-r leukemia cells. (A) Scatterplot of gene log₂ FC (VTP50469/DMSO) in 2 biological replicates of screens in OCI-AML2 (MLL-AF6) cells. (B) Boxplots of sgRNA log₂ FC (VTP50469/DMSO) for each indicated gene in OCI-AML2 cells. # denotes resistance hits identified in the screen. (C) Enrichment analysis of resistance hit genes using the Comprehensive Resource of Mammalian Protein Complexes (CORUM) protein complex database (top) and Jensen COMPARTMENTS (bottom). (D) Bar plots showing sgRNA log₂ FCs in genes with recurrent mutations in AML. The bars represent the log₂ FC for each sgRNA. The density plots above represent the distribution of all sgRNAs. (E) Competitive growth assay of mCherry-tagged sgMEN1 cells in BCOR wild-type and knockout OCI-AML2 cells. The graph shows the relative percentages of sgMEN1-expressing cells at the indicated time points after sgRNA infection. The mCherry percentage at each time point was normalized to the day 3 measurement. (F) Schematic illustrating competitive growth assays to assess growth advantage of leukemia cells expressing specific sgRNA sequences, under conditions of either DMSO or VTP50469 treatment. (G-I) Competitive proliferation assays showing the relative percentage of leukemia cells expressing EGFP-tagged sgRNA sequences under either DMSO or VTP50469 treatment in the OCI-AML2 (G; 100 nM), MV4-11 (H; 30 nM), and MOLM-13 (I; 10 nM) KMT2A-r AML cell lines. sgRNA targeting the luciferase gene sgLuc was used as a negative control. The EGFP percentage at each time point was normalized to the initial measurement. EGFP, enhanced green fluorescent protein; FC, fold change; NEG, negative control; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P <.0001.