Fig. 1: YBX1 is a pan-cancer dependency and drives cell proliferation in AML.

A Waterfall plots depicting the gene-dependency of each cold-shock protein coding gene in cell lines from the cancer dependency map portal (Achilles program, depmap.org). Cell lines are sorted by dependency rank (increasing dependency along the x axis), the CERES gene-effect score is shown on the Y axis. The red dotted lines display the arbitrary CERES score of −0.5, which is generally considered as a cutoff for a relevant gene-dependency. B Bar graphs displaying the data from a CRISPR-Cas9 cell competition assay showing the effect of deletion of each cold-shock protein family member over time. Members of the CSP-family were knocked out using single guide RNAs in murine MLL-AF9 transformed AML cells and the chimerism of knockout and wild-type cells over time is visualized in the graphs. The bars within a block of each guide RNA represent the chimerism at day 0, 3, 6, 9, 12 and 15. A decrease in the % of RFP + cells (shown on the Y axis) over time, as seen for YBX1, reflects a competitive disadvantage of cells that harbor the respective knockout. C Violin plots showing the gene-dependency of YBX1 (Achilles program, depmap.org) in all cancer types compared to all hematologic cancers and AML. Statistical analysis via unpaired t test, *p < 0.05. D Box plots showing YBX1 relative protein expression among cell lines from the cancer cell line encyclopedia (Mass-spec proteome analysis, data derived from depmap.org). Statistical analysis via unpaired t test, **p < 0.01, ***p < 0.001, ***p < 0.0001. E Immunohistochemistry for YBX1 in bone marrow of healthy donors (HD), patients with myelodysplastic syndrome (MDS) and patients with AML. Left side: representative pictures of the analyzed bone marrow histology sections (brown color: anti-YBX1). Right side: Violin-plot sowing the respective pathological scoring (Multiplied M-scores) among all specimens analyzed. Statistical analysis was performed using Mann–Whitney U Test, **p < 0.01. F Western-blot showing YBX1-protein levels in MOLM13 and OCI-AML3 cells after knockout using 3 different sgRNAs compared to empty vector control. G Bar graphs showing data from a CRISPR-Cas9 cell competition assay in MOLM13 and OCI-AML3 cells after YBX1 deletion using 3 different sgRNAs compared to knockout of RPA3 (positive control) or empty vector (negative control) at 0, 10, 20 and 30 days after starting the competition assay. H Growth curves of MOLM13 and OCI-AML3 cells after deleting YBX1 using sgRNA1 compared to non-targeting control (sgLUC) over the course of 15 days. I Flow-cytometry-based cell-cycle analysis in MOLM-13 cells after genetic inactivation of YBX1 using the BrdU assay. Left panel: representative FACS plots. Right panel: bar graphs showing quantitative analysis of cells detected in S- and G0/G1 phase. Unpaired t test, **p < 0.01, ***p < 0.001. J Flow cytometry-based assessment of CD11b surface expression of MOLM-13 and OCI-AML3 cells after knockout of YBX1 using 2 different sgRNAs. Unpaired t test, *p < 0.05, ***p < 0.001. K Representative cytological pictures showing cell morphology of MOLM-13 cells after YBX1 knockout compared to control (empty; Quick-Dip staining kit, JORVET, Loveland, CO, USA). L Apoptosis assay using Annexin V (Biolegend, San Diego, CA, USA) in MOLM-13 and OCI-AML3 cells after knockout of YBX1 using 2 different sgRNAs (day 7 and 14 after YBX1 knockout).