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Retinoblastoma, a pediatric malignancy of the retina, is primarily driven by the bi-allelic inactivation of the RB1gene. However, a subset of cases are characterized by proficient RB1 functions but with MYCN copy number mutations, suggesting an alternative oncogenic mechanism in the absence of RB1 mutations. The aim of this thesis is to investigate the intricate molecular and cellular pathways implicated in retinoblastoma, with a particular focus on the role of MYCN expression and its interplay with the cell cycle and apoptotic pathways. In Paper I, we explored the regulatory mechanisms underpinning MYCN-induced retinoblastoma using aRB1-proficient MYCN-overexpressing in vivo model in embryonic chicken retina and MYCN-transformed cells in culture. Our findings revealed that MYCN overexpression led to a significant upregulation of E2F levels, thereby dysregulating the cell cycle and mimicking the mechanistic phenotype of RB1-deficient tumors. Inhibition on E2f DNA-binding activity efficiently normalized growth and apoptosis in MYCN-transformed cells in culture. Despite RB1 proficiency, the elevated E2F levels induced a neoplastic behavior in retinal cells, indicating a novel mechanism of retinoblastoma carcinogenesis independent of RB1 inactivation. Paper II employed single-cell RNA sequencing to dissect the cellular composition of MYCN-driven retinoblastoma in chicken in vivo model, revealing a predominant origin in cone photoreceptor progenitors. This finding suggested a cell-type-specific vulnerability to MYCN-induced transformation. The research further identifies a notable heterogeneity within the MYCN-transformed cells, with a subset of cells exhibiting non-cone photoreceptor features but features of other neurons like ganglion cells. A cluster was also identified withelevated expression of genes related to malignancy and tumor progression, including UBE2C and TOP2A. This suggested a link between MYCN overexpression and tumor development, potentially mediated t

Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We studied the chicken retina, a well-established model for studying retinal neurogenesis, and generated over-expression of MYCN by in ovo electroporation. In parallel, we established an equivalent human stem cell-derived retinal organoid (retinoid) model system. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human retinoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7-9 weeks in chicken. MYCN cells could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for undifferentiated cones. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype.