CD64 expression on AML correlates with their susceptibility to DNT-mediated cytotoxicity in vitro. (A) Volcano plot of all genes enriched in live (Annexin V⁻) AML3 cells following DNT treatment. X-axis represents the log₂-transformed fold changes in normalized sgRNA tag counts in dead vs live cells. The gene targets with significantly enriched sgRNAs (FDR ≤0.10) in live cells are represented by red circles. (B-C) CRISPR screen of essential genes in the epidrug CRISPR library in remaining live AML cells vs untreated AML cells for AML2 and AML3 cells. Genes were rank-ordered by robust rank aggregation (RRA) scores calculated by MAGeCK²⁴ ; a higher RRA score indicates genes that are enriched in LIVE cells following DNT coculture (eg, genes conferring AML susceptibility to DNTs). FCGR1A is the top-ranked gene in AML2 and AML3 cell screens. (D) CD64 expression on 6 AML cell lines was analyzed by flow cytometry. Each cell line was cocultured with DNTs for 2 hours at an E:T ratio of 2:1 in triplicates. Scatterplot depicts Pearson’s correlation coefficient for the mean killing of AML against surface CD64 expression. (E) Surface expression of CD64 by flow cytometry in AML2 and AML3 Cas9-expressing cells transduced with sgRNAs against FCGR1A and a control (sgAAVS). (F-G) Cytotoxicity assays with DNTs at various E:T ratios (2 hours) in FCGR1A-deficient AML2 cells (F) and AML3 cells (G). Experiments were repeated 3 times with similar results, and statistical significance was determined by 1-way analysis of variance with the Dunnett test for multiple comparisons. ****P < .0001. MFI, mean fluorescence intensity.