Fig 2: Hypoxia-induced KDM2A and KDM2B mRNA in a HIF-α/HIF-1β-dependent manner. (A) HeLa and (B) U2OS cells were transfected with the indicated siRNA oligonucleotides for 48 h prior to RNA extraction. In addition, cells were exposed to 1% O2 for the last 24 h. Following cDNA synthesis, qPCR analysis was performed for the levels of KDM2A and KDM2B mRNA. Graphs depict mean and standard deviation from a minimum of three independent experiments performed in duplicate. Student t-test was performed and p values calculated. * p < 0.05, ** p < 0.01, *** p < 0.001. (C) HeLa and (D) U2OS cells were transfected with empty vector (EV), or HIF-1α (1 or 2 µg) for 48 h prior to RNA extraction. In addition, cells were exposed to 1% O2 for the last 24 h. Following cDNA synthesis, qPCR analysis was performed for the levels of KDM2A and KDM2B mRNA. Graphs depict mean and standard error of mean from a minimum of three independent experiments performed in duplicate. Student t-test was performed and p values calculated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: KDM2 mRNA is induced in hypoxia in human and in Drosophila melanogaster. U2OS and HeLa cells were exposed to 1% O2 for 24 h prior to RNA extraction. Following cDNA synthesis, qPCR analysis was performed for the levels of (A) KDM2A, (B) KDM2B, and (C) CA9 mRNA. Graphs depict mean and standard deviation from a minimum of three independent experiments performed in duplicate. (D) Adult animals were exposed to 5% O2 for 24 h prior to total RNA extraction. Levels of KDM2 were as in (A). Graph depicts mean and standard deviation of two independent experiments consisting of 70 animals per condition. Student t-test was performed and p values calculated. *** p < 0.001.

Fig 4: 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.

Fig 5: DNA binding and demethylase activities of KDM2A are both required for its ALT-supporting function.a Schematic of full-length KDM2A protein. The domains are labeled and color-coded. b, c CRISPR-based KDM2A tiling assay in ALT#1 (b) and Saos2 cells (c). Plotted is the fold changes of sgRNA abundance (ratio of start to endpoint) after 16 population doublings in culture. x-axis shows targeting sgRNAs and the domain location of each sgRNA within the KDM2A protein is indicated by colors; the y-axis shows the fold changes of each targeting sgRNA following the culture period. d Western blot analysis of KDM2A, Flag, and ACTB in whole-cell lysates prepared from Saos2 cells transduced with indicated constructs. The Flag-tagged wild-type and KDM2A mutants were generated from the CRISPR-resistant KDM2A-1r construct. e Competition-based proliferation assay of KDM2A-targeted sgK#1 (linked with GFP expression) in Saos2 cells transduced with the indicated constructs. The bar graphs are expressed as mean ± s.e.m. of three independent experiments; two-tailed paired t-test. f Clonogenic assay of sgK#1 in Saos2 cells transduced with the indicated CRISPR-resistant wild-type or KDM2A mutants. Crystal violet staining was conducted on day 24 post-cell seeding. Source data are provided as a Source Data file.