Fig 2: FOXR2 is sufficient in vitro and in vivo to promote proliferation and tumor formation. A, Normalized confluence of H9 hNSCs expressing FOXR2 relative to control. Values indicate mean ± SEM across three replicate experiments. **, P < 0.01, as determined by a two-tailed unpaired t test on day 7. B, Normalized spheroid size (relative to day 0) of mNSCs transduced with lentiviral vectors to induce expression of FOXR2 or vector control. Values indicate mean ± SEM across three replicate experiments. *, P < 0.05, as determined by a two-tailed unpaired t test on day 7. C, Survival curves of control (n = 13) and FOXR2-expressing (n = 10) IUE pediatric high-grade glioma (PdgfraD842V + DNp53) mouse models. ***, P < 0.0001, as determined by the log-rank Mantel–Cox test. D, Representative whole-brain images of control and FOXR2-expressing IUE pediatric high-grade glioma (PdgfraD842V + DNp53) mouse models. GFP expression indicates the cortical location of IUE-targeted pediatric high-grade glioma models. Scale bar, 1 mm. E, Representative hematoxylin and eosin (H&E) staining of control and FOXR2-expressing IUE cortical gliomas. Scale bar, 100 µm. F, Percentage of Ki67-positive cells in tumors collected from control or FOXR2-expressing IUE cortical gliomas. Values indicate mean ± SEM from tumors obtained from six mice per condition. ***, P < 0.0001, as determined by a two-tailed unpaired t test. G, Survival curves of control (n = 11) and FOXR2-expressing (n = 10) IUE brainstem DMG (PdgfraD842V + DNp53 + H3.3K27M) mouse models. ***, P < 0.0001 as determined by log-rank Mantel–Cox test. H, Representative images of control and FOXR2-expressing IUE brainstem DMG models labeled with GFP (green) and counterstained with Hoechst (blue). GFP expression indicates the brainstem location of IUE-targeted DMG models. Hoechst stains nuclei as a marker of all cells. I, Representative hematoxylin and eosin staining of control and FOXR2-expressing IUE brainstem DMG models. Scale bar, 100 µm. J, Percentage of Ki67-positive cells in tumors collected from control or FOXR2-expressing IUE brainstem DMG models. Values indicate mean ± SEM from tumors obtained from six mice per condition. ***, P < 0.0001, as determined by a two-tailed unpaired t test.

Fig 3: FOXR2 is aberrantly expressed through epigenetic mechanisms. A, Heatmap of CCLE cell lines depicting z-scores (relative to mean of methylation for each CpG site) for all CpG sites within the FOXR2 TAD on the X chromosome as a function of FOXR2 expression. FOXR2-expressing cell lines contain a 2-Mb region that is significantly hypomethylated relative to other regions of the X chromosome. FOXR2-expressing cell lines are delineated by FOXR2 expression (right). B, Quantification of FOXR2 TAD methylation score for each CCLE cancer cell line (P = 1.965e-15, as determined by Welch t test). Each cell line is represented by an individual dot, and FOXR2 expression (low vs. high) is indicated on the x-axis. C, Correlation between methylation at CpG loci and FOXR2 expression (x-axis) and the probability of these correlations (y-axis). Blue, CpG sites within the FOXR2 TAD; orange, the entire X chromosome. D, CTCF binding of the hypomethylated region shown in A that encompasses FOXR2 in FOXR2-expressing DIPG-IV diffuse midline glioma (DMG; bottom) and non–FOXR2-expressing BT245 cells (top). Three independent replicates are shown for each cell line. Two regions are highlighted: within the FOXR2 TAD (chrX: 55,320,00–55,540,000; left) and outside of the FOXR2 TAD (chrX: 64,730,000–64,830,000; right). E, Quantification of the CTCF pileup specifically in the indicated region encompassing the FOXR2 TAD in three replicates of non–FOXR2-expressing cell lines (BT245) and three replicates of FOXR2-expressing DMG cells (DIPG-IV; P = 0.0424, unpaired t test). F, Quantification of total CTCF peaks across the genome in three replicates of non–FOXR2-expressing BT245 DMG cells and three replicates of FOXR2-expressing DIPG-IV DMG cells (P = 0.1078, unpaired t test). ns, not significant.

Fig 4: FOXR2 is necessary in FOXR2-expressing cancers. A, Top three tracks: Expression and structure of FOXR2 isoforms in FOXR2-expressing DIPG-IV diffuse midline glioma (DMG) cells (green), non–FOXR2-expressing DIPG-XIII DMG cells (blue), and normal testis (black), as determined by RNA-seq. Bottom four tracks: H3K4me3 promoter and H3K27ac enhancer binding in the same FOXR2-expressing DIPG-IV DMG and non–FOXR2-expressing DIPG-XIII DMG cells. Bars below the H3K4me3 and H3K27ac ChIP-seq tracks indicate the position of statistically enriched peaks (FDR < 0.01). The position of Exon-3 is depicted with a gray bar across the different tracks.B, Firefly to Renilla luciferase relative light unit (RLU) ratio in FOXR2-expressing NCI-H838 NSCLC cells transduced with reporter constructs that harbor the Exon -3 promoter sequence relative to vector control. *, P < 0.05, as determined by a two-tailed unpaired t test comparing Exon -3 promoter sequence to control at 7 days following selection across three independent replicate experiments. C, Firefly to Renilla luciferase RLU ratio in FOXR2-expressing A375 melanoma (SKCM) cells transduced with reporter constructs that harbor the Exon -3 promoter sequence relative to vector control. *, P < 0.05, as determined by two-tailed unpaired t test comparing Exon -3 promoter sequence to control at 7 days following selection across three independent replicate experiments. D, Schematic showing the location of CRISPR interference guides (red, labeled sgRNA_1KRAB, sgRNA_2KRAB, sgRNA_3KRAB, and sgRNA_4KRAB) in relation to the FOXR2 Exon -3 promoter peak. Four CRISPR-Cas9 KO guides targeting the FOXR2 Exon +1 are also shown. E–G, Representative Western immunoblots showing FOXR2 protein levels following transduction with CRISPRi lentivirus with guides targeting different regions of the FOXR2 Exon -3 promoter peak, or nontargeting vector control using lysates derived from the following FOXR2-expressing cancer cell lines DIPG-IV DMGE (E), NCI-H838 NSCLC (F), and A375 melanoma (SKCM; G). H, Gene effect (y-axis) following FOXR2 ablation in genome-scaled CRISPR-Cas9 loss-of-function screens across 948 cancer cell lines with low vs. high levels of FOXR2 expression (x-axis). Values depict mean ± SEM. ***, P < 0.0001, as determined by two-tailed unpaired t test. I, CRISPR interference of FOXR2 using sgRNA_1-4KRAB in FOXR2-expressing DIPG-IV DMG cell line attenuates proliferation. Values indicate mean ± SEM across three replicate experiments. *, P < 0.05, as determined by two-tailed unpaired t tests comparing each guide relative to nontargeting control at 120 hours. J, Normalized confluence (relative to day 0) of FOXR2-expressing NCI-H838 NSCLC cells following transduction with CRISPRi vectors and guides targeting the Exon -3 FOXR2 promoter. Values indicate mean ± SEM across three replicate experiments. **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired t tests at 120 hours. K, Normalized confluence (relative to day 0) of FOXR2-expressing A375 melanoma (SKCM) cells following transduction with CRISPRi vectors and guides targeting the Exon -3 FOXR2 promoter. Values indicate mean ± SEM across three replicate experiments. *, P < 0.05 as determined by two-tailed unpaired t tests at 120 hours. ns, not significant.

Fig 5: ETS TFs are necessary for FOXR2-mediated proliferation. A, Normalized confluence of H9 hNSCs expressing FOXR2 + vector control, FOXR2 + ETS1sg2_KRAB, HcRed + vector control, and HcRed + ETS1sg2_KRAB. Values indicate mean ± SEM across three replicate experiments. *, P < 0.05, as determined by a two-tailed unpaired t test on day 5. B, Normalized confluence of H9 hNSCs expressing FOXR2 + vector control, FOXR2 + ETV3KRAB, HcRed + vector control, and HcRed + ETV3KRAB. Values indicate mean ± SEM across three replicate experiments. *, P < 0.05, as determined by a two-tailed unpaired t test on day 5. C, Representative Western immunoblot depicting ETS1 protein levels in H9 hNSCs expressing FOXR2 + vector control, FOXR2 + ETS1sg2_KRAB, HcRed + vector control, and HcRed + ETS1sg2_KRAB. D, Representative Western immunoblot depicting ETV3 protein levels in H9 hNSCs expressing FOXR2 + vector control, FOXR2 + ETV3KRAB, HcRed + vector control, and HcRed + ETV3KRAB. E, Schematic depicting FOXR2 protein domains. The MYC-binding domain is indicated in red and forkhead DNA-binding domain is indicated in green. F, Growth of neurospheres (measured as spheroid size) of mNSCs expressing FOXR2 WT relative to mNSCs expressing ?MYC, ?Forkhead, and control, and normalized to day 0 values. Values indicate mean ± SEM across three replicate experiments. **, P < 0.01, as determined by two-tailed unpaired t tests. G, Representative Western immunoblot depicting HA-tagged FOXR2 protein levels in mNSCs expressing FOXR2 WT, ?MYC, and ?Forkhead. H, Quantification of HA protein expression in Western immunoblots performed using lysates derived from mNSC-transduced FOXR2 WT, ?MYC, and ?Forkhead. Values from three independent experiments and their mean ± SEM are shown. ns, not significant as determined by a two-tailed unpaired t test with P > 0.05. I, Percentage of GFP-positive cells that are Ki67-positive cells in control, FOXR2 WT, ?MYC, and ?Forkhead expressing IUE tumors. Values represent Ki67% positivity in four independent tumors per condition. Black horizontal line indicates the mean across all four tumors, with error bars representing SEM. **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired t tests.