Identification and in vitro validation of RIOK2 dependency in AML. (A) Experimental overview of the kinome-wide, domain-focused CRISPR screening. Murine cells harboring an MLL-AF9 translocation were transduced with a lentiviral sgRNA library followed by puromycin selection. NGS was performed on samples immediately after and 10 days after the selection. (B) DeSeq2 analysis of the screening results. sgRNAs for positive control genes are depicted in red, negative control sgRNAs in green, and sgRNAs targeting Riok2 in purple. (C) Competition-based proliferation assays in murine MLL-AF9 and Trp53^−/− MEFs and human THP-1, BJ-TERT and MOLM13 cell lines using an sgRNA plasmid containing a BFP or GFP fluorophore. The percentage of positive cells was quantified by FACS and normalized to the nontargeting control, relative to the initial time point for each of the indicated sgRNAs. Data from a representative experiment are shown. (D) Western blots for murine and human RIOK2 4 days after sgRNA transduction. Cells were selected by puromycin for 3 days, beginning 24 hours after transduction with the indicated sgRNAs. NC-1, nontargeting control 1.