Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Oncoprint summary of mutations, copy number variations, gene expression, and gene fusions in G3 and G4 MB (n = 545 tumors). 173 samples without recurrent alterations detected are not shown. b, Gene-level summary of CBFA2T2 mutations in G4 MB. c, Structural model of CBFA2T2 protein NHR1 domain, highlighting positions affected by missense mutations. The structure of the NHR1 domain has been previously determined (Bottom), while the full protein structure was inferred using iTasser39 (Top). d, Comparison of significant focal deletions in n = 206 G4 MB, either with CBFA2T2 mutations, or PRDM6 overexpression, versus tumors without CBFA2T2 or PRDM6 abnormalities. Significance assessed using GISTIC 2.040 (FDR < 0.25).

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, Comparison, Over Expression

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Percentage of cells in normal cell types and MB cells which exhibit predicted TF activity (SCENIC44). b, OTX2 expression in the developing human RL at 11 and 18 PCW. Scale bars: 100 μm. c, OTX2 expression at 18 PCW. High expression is observed in the RL (black box) and the posterior lobes. Scale bar: 500 μm. d, Representative T1 enhanced or T2 MRI scans showing typical locations of each MB subgroup at initial diagnosis. Mid-sagittal scans are shown for G3 and G4 MB, axial scans for SHH and WNT MB tumors. e, OTX2 expression in G3 and G4 MB samples by CBFA complex alterations (Fig. 4d). Significance assessed using two-sided Mann-Whitney U test, *** p = 0.0000162, n = 545 MBs. Box plots show the median and interquartile range, and whiskers show the data range. Points outside this range are outliers and are plotted individually. f, CBFA2T2 and CBFA2T3 expression following OTX2 knockdown in G3 MB tumorspheres. Data shown as mean fold change ± SEM compared to scramble, with n = 3 biological replicates. Significance assessed using DESeq245 (FDR < 0.05), *** p < 0.0005, ** p < 0.005. CBFA2T2: HDMB03, p = 1.20e−30; MB3W1, p = 4.45e-15. CBFA2T3: HDMB03, p = 2.90e−91; MB3W1, p = 0.0055. g, Single-cells show increased similarity to normal differentiated cell types upon OTX2-KD in G3 MB tumorspheres. h, Expression of granule neuron (GN) lineage marker genes along pseudotime following OTX2-KD in MB3W1. (Top) Density of most similar developmental cell type (g) along pseudotime. (Bottom) Binned expression of GN differentiation genes (Fig.4a). Data presented in b, c were not performed in replicates.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Activity Assay, Expressing, MANN-WHITNEY, Knockdown, Marker

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, PRDM6 expression in CBFA2T2 mutant (red) and CBFA2T2 WT (grey) G3 and G4 MB samples demonstrates that enhancer hijacking mediated PRDM6 expression is largely limited to CBFA2T2 WT cases. b, Density of regions of chromosomal gain and loss along human chromosome 16q in G3 and G4 MB cases, demonstrating that deletions are biased towards the telomeric end of 16q, the location of known drivers, particularly CBFA2T3. c, CBFA2T3 expression differences between samples with and without CBFA2T3 deletions, split by subgroup. Statistical significance was assessed using Kruskal-Wallis rank-sum test (FDR < 0.05), *** p < 0.0005, G3, p = 2.88e−05; G4, p = 2.60e-09. G3, n = 112; G4, n = 206. CBFA2T3 is a copy-number responsive tumor suppressor gene in G4 MB. d, IGV analysis showing focal deleted region in two G4 MB samples MB-0364 and MB-0559. MB-0364, which is the minimal common deleted region (MCDR) on 16q in G3 and G4 MB, though does not quite achieve statistical significance in the GISTIC analysis. MB-0559 is the MCDR achieving statistical significance in GISTIC analysis. CBFA2T3 is identified with a red box. e, Cartoon illustrating the MCDR concept. f, Expression differences between copy neutral or hemizygously deleted G3 and G4 MB samples for genes within the MB-0364 MCDR on chr16q24.3. Statistical significance was assessed using two-sided Mann-Whitney U tests with FDR adjustment, * p < 0.05, *** p < 0.0005. Deletion, n = 86; Neutral, n = 232. e, Expression differences between copy neutral or hemizygously deleted G3 and G4 MB samples for genes within the MB-0559 MCDR on chr16q24.3. Statistical significance was assessed using two-sided Mann-Whitney U tests with FDR adjustment, * p < 0.05, *** p < 0.0005. Deletion, n = 86; Neutral, n = 232. A full list of p values for genes presented in (f) and (g) can be found in Supplementary Data Table 1. h, CBFA2T2 (left) and CBFA2T3 (right) expression in SHH, G3, and G4 MB by bulk RNAseq. Statistical significance was assessed by Kruskal-Wallis rank-sum test (FDR < 0.05), * p < 0.05, *** p < 0.0005. For CBFA2T2: SHH-G3, p = 2.29e−47; SHH-G4, p = 4.42e−73; G3-G4, p = 0.035. For CBFA2T3: SHH-G3, p = 7.10e−42; SHH-G4, p = 1.13e−46; G3-G4, p = 0.61. G3, n = 219; G4, n = 326; SHH, n = 250. While CBFA2T2 and CBFA2T3 are recurrently targeted and have low expression in G3 and G4 MB, high expression of both genes and an absence of alterations are observed in SHH MB. CBFA2T2 and CBFA2T3 likely have different roles in SHH MB compared to G3 and G4 MB. For c, f, g, and h box plots show the median and interquartile range, and whiskers show the data range. Points outside this range are outliers and are plotted individually.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, Mutagenesis, MANN-WHITNEY

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Dot plot showing expression of characteristic marker genes across RL glutamatergic cell types in the developing human cerebellum13. b, UMAP embeddings coloured by pseudotime inferred from Slingshot65, where the direction of pseudotime is from dark to light colours, for the granule cell lineage (Left) and the UBC lineage (Right). c, Expression of CBFA2T2 (Left) and CBFA2T3 (Right) in each zone of the developing human RL by bulk RNAseq12. Statistical significance was assessed using a two-sided Mann-Whitney U test, * p < 0.05; CBFA2T2, p = 0.0078; CBFA2T3, p = 0.0056. n = 9 biological samples, per zone, acquired between 9 and 19 PCW. Box plots show the median and interquartile range, and whiskers show the data range. Points outside this range are outliers and are plotted individually. d, UMAP embedding of 63,296 single cells derived from G3 (n = 6), G4 (n = 11), and SHH (n = 3) MB scRNAseq samples. Clusters of transcriptionally similar cells are colored and labeled by tumor sample or annotated cell type for non-tumor cells. e, Copy number variations detected in single cells inferred using inferCNV63. (Top) Reference non-tumor cells are devoid of copy number variations. (Bottom) Tumor cell clusters were enriched for copy number variations characteristic of the sample subgroup. Cells containing CNVs were assigned as tumor cells for downstream analysis. f, UMAP embedding as in (d) coloured by the detection of copy number variations. g, Dot plot showing expression of characteristic marker genes of SHH, G3, G4 MB, and the non-tumor cell types identified. For a, g, colour indicates average expression and size of each dot indicates the percent of cells in that cluster that express the genes.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, Marker, MANN-WHITNEY, Derivative Assay, Labeling

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Mutual exclusivity (ME) of somatic SNVs in 16q genes FANCA, ZFHX3, and CHD9. Deletions of 16q do not typically co-occur with mutations in these genes. G3 and G4 MB prefer deletions to simultaneously disrupt several tumor suppressor genes (TSGs). b, Locations of G4 MB TSGs on chr16 and percentage of samples where TSGs are deleted to haploinsufficiency in G3 and G4 MB samples with 16q deletions. c, ME of PRDM6 overexpression, CBFA2T2 mutations, CBFA2T3 deletions, KDM6A mutations, and GFI1 or GFI1B enhancer hijacking in G4 MB. Significance assessed using the impurity test for ME implemented by DISCOVER41. d, Cartoon of known or suspected CBFA complex members. e, Expression of significant differentially expressed genes (DEGs) between microglia from each subgroup using scRNAseq (MAST42, FDR < 0.05). f, Predicted receptor-ligand interactions between microglia and tumor cells in G4 MB (CCInx43). Node colours represent mean normalized gene expression in each cell type, and edge colour represents the average of the node expression levels. Only significant DEGs from g were included, demonstrating the microenvironment specificity of G4 MB.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Over Expression, Expressing

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Western blot showing successful expression of the TurboID-CBFA2T2 fusion protein when the TurboID construct is fused to the N-terminal of CBFA2T2, but not to the C-terminal. b, Protein-protein interaction (PPI) network of novel CBFA2T2 interacting proteins. Each node represents a protein and edges between the proteins represent known or novel PPIs. Edges in red represent known interactions between CBFA2T2 interacting proteins, and edges in green represent known interactions with CBFA2T2 that were recapitulated in our TurboID screen. Proteins are grouped with dashed lines if they contain known interactions between each other. c, Significant CBFA2T2 prey proteins enriched in each indicated biological process. GLI2 is a SHH oncogene and has been recently shown to maintain GCP proliferation and identity, implicating the CBFA complex82. d, Enrichment map of biological processes (GO:BP) enriched in CBFA2T2 prey proteins by TurboID. Each node represents a significantly enriched pathway and edges represent shared genes between nodes. Nodes are grouped and labelled with a common biological theme. Significance was assessed using G:Profiler83 with FDR correction. e, Protein-protein interaction (PPI) network of CBFA2T2 TurboID proteins and G3/G4 MB driver genes (Fig. 1a) using STRING41. Edges between CBFA2T2 and diamond-shaped nodes are not drawn for simplicity. Connectivity significance was assessed by STRING, p < 0.1e-16.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Western Blot, Expressing, Construct

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Percent of G3 and G4 MB in our cohort (n = 545) explained by alterations in genes connected to CBFA2T2 with a known or novel PPI (one step in the network presented in Extended Data Fig. 4e). Significance assessed using the impurity test for mutual exclusivity implemented in the R package DISCOVER41. b, Ranked expression of HBEGF (left) and EREG (right) in G4 MB (n = 326). Points are coloured by the presence (red) or absence (grey) of known CBFA complex alterations. Samples with the highest expression of HBEGF and EREG typically do not have CBFA complex alterations, suggesting an alternate mechanism of CBFA complex inhibition. Data presented in a were not performed in replicates. c, Summary of disrupted pathways in G3 and G4 MB. Altered genes are grouped by pathway and labelled with alteration frequency. d, (Left) H&E–stained midsagittal section from 17 PCW human cerebellum. (Right) Fluorescence immunohistochemistry (IHC) showing KI67 and SOX2 expression in the human RL compartments. Data presented in d were not performed in replicates. RLVZ and RLSVZ are denoted by red and yellow asterisks, respectively. Scale bars: 100 μm.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, Inhibition, Staining, Fluorescence, Immunohistochemistry

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Schematic summarizing Homo sapiens cerebellar development. Prior to 11 PCW, the RL resembles that of Mus musculus. Around 11 PCW the human RL splits into a ventricular and subventricular zone leading to human-specific cerebellar expansion. b, KI67 expression in the developing human RL at 11, 14 and 17 PCW. c, Percent of human RL cells expressing KI67 across several developmental timepoints. Significance assessed using a paired two-tailed t-test compared to 11 PCW, ** p < 0.005, * p < 0.05. 14PCW, p = 0.0072; 17PCW, p = 0.012; 19PCW, p = 0.0024. n = 3 biological repeats per time point; error bars, SEM. d, e, In situ hybridization (ISH) showing spatially resolved RNA expression of CBFA2T2 (d) and CBFA2T3 (e) in the developing human cerebellum at CS23, 11, and 17 PCW. CBFA2T2 and CBFA2T3 expression is first observed at 11 PCW in the RLSVZ but not the RLVZ. Data presented in b are representative images from three independent experiments with similar results, data in d and e were not performed in replicates. RLVZ, RLSVZ, and EGL are denoted by red, yellow, and black asterisks, respectively. Scale bars: 100 μm.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, Two Tailed Test, In Situ Hybridization, RNA Expression

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, (Top left) In situ hybridization (ISH) showing MKI67 expression. In-set highlights the developing cerebellum, and the RL is indicated by the black box. (Other images) Hematoxylin and eosin (H&E)–stained midsagittal sections of the developing human cerebellum. In each, the rhombic lip is indicated by the black box. Scale bars: 500 μm. b, GFAP expression in the developing human RL at 17 PCW. Scale bar: 100 μm. The RLVZ and RLSVZ are physically divided by a vascular plexus, as indicated with white asterisks. c, ISH showing spatially resolved RNA expression of HBEGF in the developing human cerebellum at 17 PCW. Scale bar: 50 μm. HBEGF foci are enriched along the RL vascular plexus. d, KI67 expression in the developing human RL at 19 PCW. Scale bar: 100 μm. e, H&E–stained midsagittal sections of the 9-month postnatal human cerebellum. Scale bar: 500 μm. The RL is only present during gestation and disappears around birth. f, g, ISH showing spatially resolved RNA expression of CBFA2T2 (f) and CBFA2T3 (g) in the developing human cerebellum at 14 PCW. Scale bars: 100 μm. h, i, ISH showing spatially resolved RNA expression of Cbfa2t2 (h) and Cbfa2t3 (i) in the developing mouse cerebellum at E15.5 (Left) and E16.5 (Right). Scale bars: 100 μm. We do not observe a similar expression pattern of either gene in the mouse RL as we do in the human RL, and note an enrichment of expression in the EGL, similar to humans. j, ISH showing spatially resolved RNA expression of LMX1A in the developing human cerebellum at 11, 14, and 17 PCW. LMX1A is highly expressed in both the RLVZ and RLSVZ, but LMX1A expression is only retained in UBCs migrating away from the RL and is completely absent in GCPs that migrate to the EGL. Data presented in d is a representative image from three independent experiments with similar results, data in remaining panels were not performed in replicates.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, In Situ Hybridization, Staining, RNA Expression

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, OTX2 ChIP-seq34 peaks are enriched at CBFA2T2 gene locus, but not CBFA2T3. b, OTX2 protein expression is reduced following OTX2-KD. Samples were used for bulk RNA sequencing. Beta actin used as a loading control. c, OTX2 protein expression is reduced following OTX2-KD. Samples were used for single-nucleus RNA sequencing. Beta actin used as a loading control. d, Representative images of primary tumorspheres in OTX2-KD and scramble conditions for both HDMB03 and MB3W1 cultures. Scale bar: 300 μm. e, f, Unbiased clustering of single nuclei following OTX2-KD in HDMB03 and MB3W1 G3 MB cells lines (c). g, Average expression of gene signatures derived from bulk RNAseq on OTX2-KD from HDMB03 and MB3W1 (b). Cells that are more orange than green indicate cells with higher expression of genes characteristic of the unchanged G3 MB cell lines, and vice-versa. Orange cells are likely cells where OTX2-KD was inefficient. h, Differentiation score as determined by CytoTRACE73. Less differentiated cells are indicated in red and more differentiated cells are indicated in blue. The results support a model where cluster 6 in HDMB03 and cluster 3 in MB3W1 represent inefficient OTX2-KD cells that retain the most similarity to WT tumor cells. i, RBFOX3 (NeuN) protein expression is increased following OTX2-KD in both HDMB03 and MB3W1, validating GN differentiation following OTX2-KD. j, Expression of genes significantly correlated with granule neuron differentiation along pseudotime in normal human RL development. (Top) Density of cell along pseudotime in the granule neuron lineage (Extended Data Fig.6b, left). (Bottom) Binned gene expression of markers derived from the developing human cerebellum snRNAseq dataset (Fig.4a). The stepwise expression of granule neuron genes observed when OTX2 is knocked down in G3 MB (Fig.5i) strikingly mirrors that of normal granule neuron differentiation, suggesting that G3 and G4 MB arise from failed normal differentiation rather than alternate hypotheses, such as trans- or de-differentiation. k, CBFA2T2 and CBFA2T3 expression in HDMB03 (Left) and MB3W1 (Right). CBFA2T2 expression is strongly upregulated in cells where the CytoTRACE score drops below 0.8, and CBFA2T3 follows. The results suggest CBFA2T2 and then CBFA2T3 are upregulated early in response to efficient OTX2-KD. l, CBFA2T2 expression change in response to CBFA2T2 overexpression (CBFA2T2-OE) in HDMB03 by qPCR. m, CBFA2T2 protein expression is increased following CBFA2T2-OE. β-actin used as a loading control. n, Representative images of primary tumorspheres in CBFA2T2-OE GFP and Control RFP conditions. Scale bar: 600 μm. o, p, Live cell number (o) and viability (p) in response to CBFA2T2-OE. Live cell number is significantly reduced in response to CBFA2T2-OE, while viability is unchanged. Data are normalized to their respective controls and presented showing points from n = 3 technical replicates per N = 8 or N = 5 biological replicates, for live cell number and viability, respectively. Error bars indicate SEM. Significance assessed using a two-tailed paired t-test on biological replicates, ** p = 0.0047. q, OTX2 restrains differentiation of RL progenitors through CBFA complex inhibition. Data presented in d, n are representative images from 4 and 8 independent experiments, respectively, with similar results, data in b, c, i, m were not performed in replicates. For gel source data, see Supplementary Figure 1.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing, RNA Sequencing Assay, Control, Derivative Assay, Over Expression, Two Tailed Test, Inhibition

Journal: Nature

Article Title: Failure of human rhombic lip differentiation underlies medulloblastoma formation

doi: 10.1038/s41586-022-05215-w

Figure Lengend Snippet: a, Scaled OTX2, CBFA2T2, and CBFA2T3 expression by scRNAseq. b, Expression of OTX2 at 14 and 17 PCW by ISH in the developing human cerebellum. c, T1 enhanced or T2 mid-sagittal MRI images of G4 MB (n = 12) tumors at initial diagnosis. d, T1 enhanced or T2 mid-sagittal MRI images of G3 MB (n = 10) tumors at initial diagnosis. Both G3 and G4 MB tumors present exclusively in the OTX2+ inferior cerebellum. e, Axial T1 enhanced, T2 or FLAIR images of SHH MB (n = 3) at initial diagnosis. SHH tumors occur in the cerebellar hemispheres, consistent with an EGL cell of origin. f, Axial T1 enhanced, T2 or FLAIR images of WNT MB (n = 3) at initial diagnosis. Data presented in b were not performed in replicates.

Article Snippet: 3xHA-TurboID-NLS_pCDNA3 was a gift from Dr. Alice Ting (Addgene plasmid # 107171). pCMV6-AC-CBFA2T2-GFP was purchased from Origene (RG202013).

Techniques: Expressing