CRISPR screen of the ACER library identifies crucial epigenetic regulators of menin inhibitor response. (A) Schematic of ACER library design. Classical epigenetic modulators (n = 333) were extended by including 343 interactors from BioPlex Interactome and 102 codependency genes from the DepMap data set, resulting in a collection of 758 epigenetic regulator genes. (B) Schematic overview of the CRISPR knockout screen using the ACER library. OCI-AML2 AML cells with stable Cas9 expression were transduced with pooled sgRNAs using lentiviruses. After antibiotic selection, these cells were treated with DMSO, 1 μM VTP50469, or 100 nM venetoclax for 0, 7, and 14 days. After treatment, the genomic DNA was harvested for library construction and analyzed using next-generation sequencing. (C) Principal component analysis showing time-dependent yet distinct cellular responses of the DMSO, VTP50469, and venetoclax treatment groups. (D-F) MAGeCK analysis results showing a ranking of genes based on their RRA scores. These rankings compare the day 14 DMSO-treated group with the day 0 DMSO-treated group (D), the day 14 VTP50469-treated group with the day 14 DMSO-treated group (E), and the day 14 venetoclax-treated group with the day 14 DMSO-treated group (F). Screen hits that were significantly dropped and enriched (RRA < 0.0001) are marked in blue and red, respectively, with the gene names of the top hits labeled for reference. BAH, bromo-adjacent homology; Bromo, bromodomain; Cas9, CRISPR-associated protein 9; CHROMO, chromatin organization modifier; DNA-DMs, DNA demethylases; DNMTs, DNA methyltransferases; HATs, histone acetyltransferases; HDACs, histone deacetylases; KMTs, lysine methyltransferases; KDMs, lysine demethylases; MBD, methyl-CpG binding domain; MOI, multiplicity of infection; PC, polycomb; PHD, plant homeodomain; PRMTs, protein arginine methyltransferases; PWWP, proline-tryptophan-tryptophan-proline domain; RNA-DMs, RNA demethylases; RNMTs, RNA methylrtansferases; RRA, robust rank aggregation.