Fig. 1: CRISPR-based screens identify KDM2A as selectively essential in ALT-dependent cells.

a Western blot analysis of ATRX protein expression in whole-cell lysates prepared from the indicated cells. b Ranking of sgRNAs by log2 fold-change (log2FC) of abundance (ratio of start to endpoint) in ALT#1 cells. the x-axis shows targeting sgRNAs; the y-axis shows the log₂FC of each targeting sgRNA after 16 population doublings. c Gene dependency scores (GDS) in IMR90-T (x-axis) versus ALT#1, ALT#2, or ALT#3 cells (y-axis). The GDS was calculated by averaging the log2FC of all sgRNAs targeting that gene. d Heatmap depicts log2FC of average sgRNA abundance of selected genes in indicated cells after 16 population doublings. e, f Competition-based proliferation assay of KDM2A-targeted sgK#1 and sgK#2 in ALT#1 (e) or Cas9-expressing Saos2 cells (f). A GFP reporter is linked to sgRNA expression. Plotted is the %GFP cells (normalized to the d0 measurement) at the indicated time points. The non-targeting sgCtrl was included as a negative and sgRNA targeting PCNA as a positive control. g, h Clonogenic assay (g) and KDM2A western blot analysis (h) of sgCtrl, sgK#1, or sgK#2-transduced ALT#1 cells. Crystal violet staining was conducted on day 20 post-seeding. i, j Crystal violet-based clonogenic survival assay (i) and KDM2A western blot analysis (j) of sgCtrl, sgK#1, or sgK#2-transduced Saos2 cells. Crystal violet staining was conducted on day 24 post-seeding. k Competition-based proliferation assay of GFP-linked sgRNA in Saos2 cells complemented with either empty vector control or the CRISPR-resistant KDM2A synonymous mutants (KDM2A-1r or KDM2A-2r). Note, the KDM2A-1r-transduced cells are resistant to sgK#1 but sensitive to sgK#2 action; the KDM2A-2r-transduced cells are resistant to sgK#2 but sensitive to sgK#1 action. l Tumor growth curves of sgCtrl, sgK#1 or sgK#2-transduced Saos2 cells. Data were expressed as mean ± s.e.m. of six biological replicates; two-tailed unpaired t-test. m Image of tumors collected at week 8 post subcutaneous transplantation. In (e, f, k, data were expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t-test. Source data are provided as a Source Data file.