Abstract
Medulloblastoma is one of the most common malignant embryonal brain tumors in children. Medulloblastomas of the Sonic Hedgehog (SHH) group arise from excessive proliferation of granule neuron progenitor (GNP) cells during cerebellar development. Genetic predisposition accounts for nearly 40% of all pediatric SHH-medulloblastomas. Recently, ELP1, a novel predisposition gene, was shown to be germline mutated in 15% of SHH-medulloblastoma patients. ELP1 encodes the scaffolding member of the Elongator complex and is required for efficient translation. Heterozygous mutations in ELP1 have been associated with the neural disorder Familial Dysautonomia, but not cancer. ELP1-associated medulloblastomas frequently harbor somatic PTCH1 co-mutations. It remains unclear how ELP1 affects the GNP lineage during normal cerebellar development and tumorigenesis in pediatric SHH-medulloblastoma patients. To characterize ELP1 mutations in the GNP lineage in vitro, we established a cerebellar organoid model from human induced pluripotent stem cells (iPSCs). We genetically inserted an EGFP reporter downstream of the endogenous GNP-specific ATOH1 locus in control iPSCs and generated cerebellar organoids according to published protocols. Marker gene and protein expression levels confirmed the cerebellar identity of the 3D model. Furthermore, activation of the EGFP reporter in single cells within the organoid highlighted the specification of putative GNPs. Next, we will determine the specific cell state of putative iGNPs and compare to human GNPs identified in our scRNAseq cerebellum atlas. To analyze tumorigenesis through ELP1 loss, we will introduce patient-specific ELP1mutations into ATOH1-EGFP iPSCs. Cerebellar organoids derived from ELP1-, PTCH1-deficient and control iPSCs will serve as models to study GNP proliferation, differentiation, apoptosis and tumor formation. Combining genome editing, in vitro 3D differentiation and functional studies, we will characterize the novel predisposition gene ELP1in GNPs during cerebellar development. In addition, we will determine the interplay of ELP1and PTCH1 co-mutations, predisposing SHH-medulloblastoma formation.