Fig. 1: CRISPR-Cas9 library screening identifies a role for FOXK1, FOXO1, KEAP1, and PTEN genes, and mitochondrial damage and ROS production in CDC.

A Schematic overview of the CRISPR-library screen, performed in triplicate using TKOv3-in-pLCKO lentiviral library in the CDC-sensitive, Cas9-expressing DLBCL cell line RI-1. Post-screening, CD37⁺ cells were sorted and processed for gDNA isolation, sgRNA-enrichment, and sequencing. B Top 15 gene hits from the screen in CD37⁺ cells, identified by DrugZ-analysis and ranked by median count per gene. C CD37 surface expression (median fluorescence intensity; MFI) and D DuoHexaBody-CD37-induced CDC (%; at 0.5 µg/mL) in RI-1 cells and the CRISPR-targeted FOXK1, FOXO1, KEAP1, and PTEN variants (n ≥ 4; data shown as mean ± SD). E Detection of dysfunctional mitochondria in RI-1 cells after 5 min of DuoHexaBody-CD37 (0.5 µg/mL) CDC induction, defined by maintained mitochondrial mass (MitoTracker Green; MTG) and decreased membrane potential (MitoTracker DeepRed; MTDR), compared to no antibody control. Representative dot-plots are shown (n = 3). F Fold-change mitochondrial ROS (mtROS) levels upon 5 min of incubation with DuoHexaBody-CD37 (0.5 µg/mL) in CDC-assays in RI-1 cells, measured by MitoSOX (MFI) in functional and dysfunctional mitochondria, and normalized to no antibody control (n = 3; mean ± SD). G DuoHexaBody-CD37-induced CDC (%; at 0.5 µg/mL) in RI-1 cells after 2-h pre-treatment with ROS-scavenger n-acetylcysteine (NAC; 10 and 20 µM) (n = 4; mean ± SD). H Detection of dysfunctional mitochondria and I fold-change mtROS induction in U2932 cells upon 5 min CDC assay with 1 µg/mL DuoHexaBody-CD37 (n = 3). Representative dot-plot of dysfunctional mitochondria detection is shown. J Effect of NAC pre-treatment on DuoHexaBody-37-induced CDC in U2932 cells (n = 3). K Fold-change in mtROS levels in dysfunctional mitochondria upon 5 min CDC assay with DuoHexaBody-CD37 (0.5 µg/mL) in RI-1 cells and gene-targeted variants (n = 3). Data are shown in a Box and Whiskers plot. N indicates independent experiments, each performed in replicate or as triplicates; the average value per experiment was used for analysis. Statistical analyses were performed using a one-way ANOVA with post-hoc Tukey’s multiple comparison test.