Gfi1 is required for tumor maintenance. a Constructs used to generate MG^flox tumors. NSCs from a CAG-CreER^TM mouse were transduced with viruses encoding Gfi1^flox-IRES-GFP and Myc^T58A-IRES-Luciferase. b Bioluminescence imaging and whole mount fluorescence image of representative MG^flox tumor. c Western blot for Gfi1 protein in MG^flox tumor cells treated overnight with vehicle control (DMSO) or 5 µM 4-hydroxytamoxifen (4-OHT). d Bioluminescence imaging of mice transplanted with cells treated with vehicle or 4-OHT. X’s denote animals euthanized before they could be imaged. e Survival curves from a representative experiment (control n = 23, 4-OHT n = 24). p Value < 0.0001 was determined by Log-rank (Mantel–Cox) test. f Western blot of Gfi1 protein in resulting MG tumors from vehicle and 4-OHT treatment groups

Gfi1 is associated with Lsd1. a–c Co-immunoprecipitations of a Gfi1, b Lsd1, and c CoREST were performed on MG tumor cells, and Gfi1, Lsd1, and CoREST protein levels were detected by Western blotting. Input represents 10% of total lysate before immunoprecipitation with experimental and isotype control antibodies

The SNAG domain is required for Gfi1-driven tumorigenesis. a Structure of Gfi1, illustrating the N-terminal SNAG domain and six C-terminal zinc fingers. Arrow denotes the proline to alanine point mutation of the SNAG mutant. b Western blot for Gfi1 protein levels in 293T cells transduced with empty vector, Gfi1-WT, or Gfi1-P2A. c qPCR for Gfi1 mRNA levels in NSCs transduced with empty vector, Gfi1-WT, or Gfi1-P2A. Data shown are from one representative experiment, where experiments were repeated in at least three biological replicates. Error bars represent 95% confidence intervals calculated using the sum of squares method. d Bioluminescence imaging of mice transplanted with NSCs that were co-infected with viruses encoding Myc and Gfi1-WT (MG) or Myc and Gfi1-P2A (MG-P2A) overnight. X’s denote animals euthanized before they could be imaged. e Survival curves comparing mice transplanted with MG or MG-P2A cells (MG n = 6, MG-P2A n = 6). p Value = 0.0005 was determined by Log-rank (Mantel–Cox) test

Genetic deletion of Lsd1 impairs growth of Gfi1-driven MB. a Western blot for Lsd1 protein in Lsd1-iKO tumor cells that were treated overnight with vehicle control (DMSO) or 5 µM 4-hydroxytamoxifen (4-OHT). b Bioluminescence imaging of mice transplanted with Lsd1-iKO cells treated with vehicle or 4-OHT. X’s denote animals euthanized before they could be imaged. c Survival curves from a representative experiment (control n = 10, 4-OHT n = 10). p Value < 0.0001 was determined by Log-rank (Mantel–Cox) test. d Western blot for Lsd1 protein in tumors from vehicle and 4-OHT treatment groups. e In vitro proliferation assay for Lsd1-iKO MG tumor cells treated with vehicle (DMSO), 1 or 5 µM 4-OHT. Proliferation was measured via ³H-thymidine incorporation at 48 h. Data are from a representative experiment and are plotted as the means of technical triplicate samples ± SEM. Experiments were repeated in at least three biological replicates. f Quantification of Ki67 staining in Lsd1-iKO MG cells after treatment with vehicle or 5 µM 4-OHT. p Value = 0.0047, t = 10.27, df = 2, one-tailed paired t test. g Quantification of active Caspase-3 staining in Lsd1-iKO MG cells after treatment with vehicle or 5 µM 4-OHT. p Value = 0.0286, t = 4.003, df = 2, one-tailed paired t test. f, g Staining and flow cytometric analysis for Ki67 and Caspase 3 were performed on fixed and permeabilized cells. Resulting values were normalized to vehicle control, and data shown represent the means of three biological replicates ± SEM

The p53 pathway remains functional in Gfi1-driven tumors. a, b Western blots for p53 and p21 protein in wild-type mouse embryonic fibroblasts (MEFs) after 4-h treatment with a 0, 0.1, or 0.5 μM doxorubicin or b 0, 2, 4, or 8 Gy of γ-irradiation. c, d Western blots for p53 and p21 protein in MG tumor cells after 4-h treatment with c doxorubicin or d γ-irradiation. e, f Western blots for p53 and p21 protein in MG tumor cells expressing DNp53 after 4-h treatment with e doxorubicin or f γ-irradiation

Neuronal commitment and differentiation pathways are downregulated in MG tumors. a Volcano plot of of 21,304 genes whose expression was compared in MG tumors (n = 7) and NSCs (n = 5). Red indicates genes that were significantly upregulated in MG (log₂FC > 1.5 and FDR-corrected p value < 10⁻²) and green indicates those that were significantly downregulated in MG (log₂FC < −1.5 and FDR-corrected p value < 10⁻²). b Pathway analysis of differentially expressed genes. Each node represents a GO term. Node size reflects the enrichment significance of the term. Edges represent association strength between the terms calculated using chance corrected kappa statistics (a standard kappa score level threshold was used). c Heat maps comparing expression of genes involved in central nervous system neuron differentiation (left), neuron fate commitment (center), and neuron migration (right) in MG tumor cells and NSCs. d Fbxo5 expression from microarray analysis comparing NSCs to MG tumor cells (left) and qPCR validation of Fbxo5 mRNA expression in MG tumor samples compared to NSC samples (right). p values < 0.0001 were determined by one-sided Welch Two Sample t test. Whiskers indicate minimum and maximum expression values, bottom of boxes indicate first quartiles, midline indicates median expression values, and top of boxes indicate third quartiles. e Bioluminescence imaging of mice transplanted with cells infected with empty vector control (GFP) or Fbxo5. X’s denote animals euthanized before they could be imaged. f Survival curves comparing mice transplanted with cells infected with empty vector (GFP) or Fbxo5 (GFP n = 28, Fbxo5 n = 27). p Value < 0.0001 was determined by Log-rank (Mantel–Cox) test. g FBXO5 expression in human Group 3/Group 4 MB samples with or without GFI1/GFI1B activation (GFI1/GFI1B n = 17, WT n = 130). p Value was determined by one-sided Welch Two Sample t test. Whiskers indicate minimum and maximum expression values, bottom of boxes indicate first quartiles, midline indicates median expression values, and top of boxes indicate third quartiles

Pharmacological inhibitors of Lsd1 inhibit growth of Gfi1-driven MB. a, b In vitro proliferation assays for MG and MP tumor cells treated with a GSK-LSD1 or b ORY-1001. Proliferation was measured via ³H-thymidine incorporation at 48hrs. c Cell viability of post-mitotic granule neurons treated with GSK-LSD1 (blue) or ORY-1001 (purple). Viability was measured via CellTiter-Glo Luminescent Assay at 48 h. Data shown in a–c are from representative experiments and are plotted as the means of technical triplicate samples ± SEM. All experiments were repeated in at least three biological replicates. d–h Mice implanted with subcutaneous MG tumors underwent surgical resection of tumors and were subsequently treated with vehicle (saline) or 10 mg/kg GSK-LSD1 in cycles of four days on and three days off (vehicle n = 21, GSK-LSD1 n = 22). d Representative images of two tumors before (left) and after (right) surgical resection. Tumors are outlined by white dotted lines. Tumor growth was monitored weekly by e bioluminescent imaging and f caliper measurements. Red arrowhead indicates time of surgical resection. Blue arrowhead indicates start of drug treatment. Data shown are from a representative experiment and are plotted as the means ± SEM. When tumors reached the maximum allowed diameter, mice were sacrificed and resulting tumors were g collected and h weighed. Compared to resection alone, resection and GSK-LSD1 treatment significantly reduced tumor burden in mice. p < 0.0001, t = 6.042, df = 41, one-tailed unpaired t test. Data shown in h are from three replicate experiments and plotted as the means ± SD