Human Retinal Organoid Modeling Defines Developmental Window and Therapeutic Vulnerabilities in MYCN-Amplified Retinoblastoma

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however, its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here, we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids, revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation, expressed SOX2, and lacked CRX, consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas, particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities, demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1, Flavopiridol) and the cell-cycle inhibitor Volasertib, indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively, our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma, establishes a robust and clinically relevant human retinal organoid platform, and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype.