Fig 2: CBL0137 decreases the transcription of NEUROD1 and OTX2-associated genes.A, B Volcano plot of the normalized expression of genes in MYC-amplified medulloblastoma cells after CBL0137 treatment. Red points represent genes with P-value <0.05. C NEUROD1 expression in the MYC-amplified medulloblastoma cells which were treated with CBL0137 or DMSO was measured using RT-qPCR. D–G Enrichment plots of genes in shOTX2_48 h and shOTX2_96 h gene sets in DMSO-treated cells and CBL0137-treated cells. H ChIP-qPCR analysis of SSRP1 levels at MYC in MB cells treated with DMSO or CBL0137. The seven sites at the promoter region of NEUROD1 ware arranged up the histogram. (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 3: CBL0137 suppresses MYC and MYC-dependent transcription.A The top pathways obtained from hallmark pathway analysis affected by CBL0137 treatment in D458 and HDMB03 cell lines. Bars represent significance of gene enrichment for a given pathway on a –log10(P value) scale. B Enrichment plots from GSEA for the MYC_TARGETS gene sets in medulloblastoma cells which were treated with DMSO or CBL0137. C MYC expression in the MYC-amplified medulloblastoma cells treated with CBL0137 or DMSO was investigated using RT-qPCR. (*P < 0.05, **P < 0.01, and ***P < 0.001). D MYC expression in the MYC-amplified medulloblastoma cells treated with CBL0137 or DMSO was investigated using immunoblotting. E Immunoblotting analyses of MYC protein expression in the xenograft tumors derived from control and CBL0137-treated cells. F Immunohistochemical staining of MYC in vehicle or CBL0137-treated intracranial orthotopic tumors (scale bar: 50 µM). G ChIP-qPCR analysis of SSRP1 levels at MYC in MB cells treated with DMSO or CBL0137. The 12 sites at the promoter region of MYC ware arranged up the histogram. (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 4: Identification of DTX2 substrates.(A) Volcano plot displaying proteins that bind to Myc-DTX2 versus EV. Myc-trap pull-downs were performed in triplicate on whole-cell lysates from HEK293 cells expressing Myc-DTX2 or EV. Proteins shown as blue dots were identified as significantly enriched proteins using a t test with a 5% false discovery rate (FDR) in all three replicates and more abundant in Myc-DTX2 pull-downs compared to EV. Associated with data S1. (B) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 or EV in the presence and absence of ABT-888 using anti-PARP1, anti-SPT16, anti-SSRP1, anti-XRCC5, anti-XRCC6, anti-RPA1, anti–Myc-tag, or anti-actin as indicated. (C) Volcano plot displaying log2 fold change in intensities in peptides modified with Ub from lysates from HEK293 cells expressing GFP-DTX2 compared to EV. Assays were performed in triplicate, and significant changes in ubiquitinated peptide intensities were determined using a Student’s t test with a 1% FDR (permutation-based). Significantly changed ubiquitinated peptides present in all three replicates, more abundant in lysates from cells expressing GFP-DTX2, and from proteins annotated in the Gene Ontology database with the term “DNA repair” are colored as indicated. Associated with data S2. (D) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, EV or Myc-DTX2, and GFP–histone H3.1 (right only) in the presence and absence of ABT-888. The cell lysates and Ni2+–pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. (E) Reduced in vitro autoubiquitination assay showing the formation of GST-2RD-IR-Ubn over time in the presence and absence of ABT-888. IR-Ub, Ub labeled with near-infrared dye. Visualized with the Odyssey CLx Imaging System (LI-COR Biosciences, top) and subsequently by staining with InstantBlue (Expedeon, bottom).

Fig 5: Integrated screen establishes SSRP1 as the top drug candidate and a prognostic marker.A Flowchart of bioinformatics analysis. B Genes with inhibitor were chosen for further validation. C Box plots of SSRP1 expression levels in different subgroups of medulloblastoma and normal cerebellum (CB) from the Pomeroy dataset. D Box plots of SSRP1 expression levels in medulloblastoma and normal cerebellum from the u133p2 dataset. E Box plots of SSRP1 expression levels in various subgroups of medulloblastoma from the Cavalli dataset. F, G Kaplan–Meier plots that show the OS rate in Cavalli dataset of all MB patients and group three MB patients. (*P < 0.05, **P < 0.01, and ***P < 0.001).