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Novel mutations target distinct subgroups of medulloblastoma.

Robinson G, Parker M, Kranenburg TA, Lu C, Chen X, Ding L, Phoenix TN, Hedlund E, Wei L, Zhu X, Chalhoub N, Baker SJ, Huether R, Kriwacki R, Curley N, Thiruvenkatam R, Wang J, Wu G, Rusch M, Hong X, Becksfort J, Gupta P, Ma J, Easton J, Vadodaria B, Onar-Thomas A, Lin T, Li S, Pounds S, Paugh S, Zhao D, Kawauchi D, Roussel MF, Finkelstein D, Ellison DW, Lau CC, Bouffet E, Hassall T, Gururangan S, Cohn R, Fulton RS, Fulton LL, Dooling DJ, Ochoa K, Gajjar A, Mardis ER, Wilson RK, Downing JR, Zhang J, Gilbertson RJ - Nature (2012)

Bottom Line: Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups.Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood.Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: St Jude Children's Research Hospital, Washington University Pediatric Cancer Genome Project, Memphis, Tennessee 38105, USA.

ABSTRACT
Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

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Genes mutated in WNT-subgroup medulloblastomas regulate LRLPs(a) Isolated Olig3+/Wnt1+ LRLPs were transduced in (b) with mutant Ctnnb1 (above hashed line) or the indicated shRNA-Red Fluorescence Protein construct (below hashed line). LRLPs were also transduced (+) or not (−) with a Tcf-Lef-enhanced green fluorescence (Tcf) reporter. Numbers right report clonal % 2′ to 3′ passage neurosphere formation (+SD). (c) Knock-down of genes targeted by shRNA relative to control transduced cells. (d) Immunofluorescence of P1 mouse hindbrains electroporated in utero at E14.5 with GFP (to control for equivalence of electroporation between embryos control) and the indicated construct. High-power views of indicated areas are shown right. Cells targeted by Ddx3x-shRNA are present 48 hours post electroporation but ablated by P1. Scale=200μm. (e) Heatmap reporting the distribution of GFP+/RFP+ cells in eletroporated mice at P1. Median distance migrated by cells, and p-values of migration distance and cell number relative to controls is shown (****, p<0.00005; ***, p<0.0005; **, p<0.005; *, p<0.05. Red and green text reports significant increase or decrease, respectively relative to control).
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Figure 3: Genes mutated in WNT-subgroup medulloblastomas regulate LRLPs(a) Isolated Olig3+/Wnt1+ LRLPs were transduced in (b) with mutant Ctnnb1 (above hashed line) or the indicated shRNA-Red Fluorescence Protein construct (below hashed line). LRLPs were also transduced (+) or not (−) with a Tcf-Lef-enhanced green fluorescence (Tcf) reporter. Numbers right report clonal % 2′ to 3′ passage neurosphere formation (+SD). (c) Knock-down of genes targeted by shRNA relative to control transduced cells. (d) Immunofluorescence of P1 mouse hindbrains electroporated in utero at E14.5 with GFP (to control for equivalence of electroporation between embryos control) and the indicated construct. High-power views of indicated areas are shown right. Cells targeted by Ddx3x-shRNA are present 48 hours post electroporation but ablated by P1. Scale=200μm. (e) Heatmap reporting the distribution of GFP+/RFP+ cells in eletroporated mice at P1. Median distance migrated by cells, and p-values of migration distance and cell number relative to controls is shown (****, p<0.00005; ***, p<0.0005; **, p<0.005; *, p<0.05. Red and green text reports significant increase or decrease, respectively relative to control).

Mentions: We showed previously that mutant Ctnnb1 initiates WNT-subgroup medulloblastoma by arresting the migration of LRLPs from the embryonic dorsal brainstem to the pontine grey nucleus (PGN)5. Therefore, to test if disruption of CDH1 might substitute for mutant CTNNB1 in medulloblastoma, we used shRNAs to knock down Cdh1 in embryonic day (E) 14.5 mouse LRLPs (Figure 3a to c). Deletion of Cdh1 expression upregulated Tcf/Lef mediated gene transcription in LRLPs and more than doubled their self-renewal capacity (Figure 3b). Furthermore, in utero electroporation of LRLPs with Cdh1 shRNAs impeded their migration from the dorsal brainstem to the PGN with an efficiency similar to that of mutant Ctnnb1 (Figure 3d,e; see Supplementary Methods). These data support the hypothesis that CDH1 suppresses the formation of WNT-subgroup medulloblastoma by regulating WNT-signals in LRLPs.


Novel mutations target distinct subgroups of medulloblastoma.

Robinson G, Parker M, Kranenburg TA, Lu C, Chen X, Ding L, Phoenix TN, Hedlund E, Wei L, Zhu X, Chalhoub N, Baker SJ, Huether R, Kriwacki R, Curley N, Thiruvenkatam R, Wang J, Wu G, Rusch M, Hong X, Becksfort J, Gupta P, Ma J, Easton J, Vadodaria B, Onar-Thomas A, Lin T, Li S, Pounds S, Paugh S, Zhao D, Kawauchi D, Roussel MF, Finkelstein D, Ellison DW, Lau CC, Bouffet E, Hassall T, Gururangan S, Cohn R, Fulton RS, Fulton LL, Dooling DJ, Ochoa K, Gajjar A, Mardis ER, Wilson RK, Downing JR, Zhang J, Gilbertson RJ - Nature (2012)

Genes mutated in WNT-subgroup medulloblastomas regulate LRLPs(a) Isolated Olig3+/Wnt1+ LRLPs were transduced in (b) with mutant Ctnnb1 (above hashed line) or the indicated shRNA-Red Fluorescence Protein construct (below hashed line). LRLPs were also transduced (+) or not (−) with a Tcf-Lef-enhanced green fluorescence (Tcf) reporter. Numbers right report clonal % 2′ to 3′ passage neurosphere formation (+SD). (c) Knock-down of genes targeted by shRNA relative to control transduced cells. (d) Immunofluorescence of P1 mouse hindbrains electroporated in utero at E14.5 with GFP (to control for equivalence of electroporation between embryos control) and the indicated construct. High-power views of indicated areas are shown right. Cells targeted by Ddx3x-shRNA are present 48 hours post electroporation but ablated by P1. Scale=200μm. (e) Heatmap reporting the distribution of GFP+/RFP+ cells in eletroporated mice at P1. Median distance migrated by cells, and p-values of migration distance and cell number relative to controls is shown (****, p<0.00005; ***, p<0.0005; **, p<0.005; *, p<0.05. Red and green text reports significant increase or decrease, respectively relative to control).
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Related In: Results  -  Collection

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Figure 3: Genes mutated in WNT-subgroup medulloblastomas regulate LRLPs(a) Isolated Olig3+/Wnt1+ LRLPs were transduced in (b) with mutant Ctnnb1 (above hashed line) or the indicated shRNA-Red Fluorescence Protein construct (below hashed line). LRLPs were also transduced (+) or not (−) with a Tcf-Lef-enhanced green fluorescence (Tcf) reporter. Numbers right report clonal % 2′ to 3′ passage neurosphere formation (+SD). (c) Knock-down of genes targeted by shRNA relative to control transduced cells. (d) Immunofluorescence of P1 mouse hindbrains electroporated in utero at E14.5 with GFP (to control for equivalence of electroporation between embryos control) and the indicated construct. High-power views of indicated areas are shown right. Cells targeted by Ddx3x-shRNA are present 48 hours post electroporation but ablated by P1. Scale=200μm. (e) Heatmap reporting the distribution of GFP+/RFP+ cells in eletroporated mice at P1. Median distance migrated by cells, and p-values of migration distance and cell number relative to controls is shown (****, p<0.00005; ***, p<0.0005; **, p<0.005; *, p<0.05. Red and green text reports significant increase or decrease, respectively relative to control).
Mentions: We showed previously that mutant Ctnnb1 initiates WNT-subgroup medulloblastoma by arresting the migration of LRLPs from the embryonic dorsal brainstem to the pontine grey nucleus (PGN)5. Therefore, to test if disruption of CDH1 might substitute for mutant CTNNB1 in medulloblastoma, we used shRNAs to knock down Cdh1 in embryonic day (E) 14.5 mouse LRLPs (Figure 3a to c). Deletion of Cdh1 expression upregulated Tcf/Lef mediated gene transcription in LRLPs and more than doubled their self-renewal capacity (Figure 3b). Furthermore, in utero electroporation of LRLPs with Cdh1 shRNAs impeded their migration from the dorsal brainstem to the PGN with an efficiency similar to that of mutant Ctnnb1 (Figure 3d,e; see Supplementary Methods). These data support the hypothesis that CDH1 suppresses the formation of WNT-subgroup medulloblastoma by regulating WNT-signals in LRLPs.

Bottom Line: Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups.Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood.Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: St Jude Children's Research Hospital, Washington University Pediatric Cancer Genome Project, Memphis, Tennessee 38105, USA.

ABSTRACT
Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

Show MeSH
Related in: MedlinePlus