Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum.

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma.

The developmental stage of the medulloblastoma cell-of-origin restricts Sonic hedgehog pathway usage and drug sensitivity.

Sonic hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage, which depends on Hedgehog signaling for its perinatal expansion. Whereas SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate whether the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as a function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream SHH pathway component Smoothened, whereas sensitivity to downstream components SUFU and GLI family proteins was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients.

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma.

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

CREB signaling activity correlates with differentiation and survival in medulloblastoma.

While there has been significant progress in the molecular characterization of the childhood brain cancer medulloblastoma, the tumor proteome remains less explored. However, it is important to obtain a complete understanding of medulloblastoma protein biology, since interactions between proteins represent potential new drug targets. Using previously generated phosphoprotein signaling-profiles of a large cohort of primary medulloblastoma, we discovered that phosphorylation of transcription factor CREB strongly correlates with medulloblastoma survival and associates with a differentiation phenotype. We further found that during normal cerebellar development, phosphorylated CREB was selectively expressed in differentiating cerebellar granule neuron progenitor (CGNP) cells. In line, we observed increased differentiation in CGNPs treated with Forskolin, Bmp6 and Bmp12 (Gdf7), which induce CREB phosphorylation. Lastly, we demonstrated that inducing CREB activation via PKA-mediated CREB signaling, but not Bmp/MEK/ERK mediated signalling, enhances medulloblastoma cell sensitivity to chemotherapy.