An immature, dedifferentiated, and lineage-deconstrained cone precursor origin of N-Myc-initiated retinoblastoma.

Abstract

Most retinoblastomas develop from maturing cone precursors in response to biallelic <i>RB1</i> loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack <i>RB1</i> mutations but have <i>MYCN</i> amplification (<i>MYCN^A</i>), N-Myc protein overexpression, and more rapid and invasive growth, yet the <i>MYCN^A</i> retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to <i>RB1</i> loss, both immature cone arrestin-negative (ARR3^-) and maturing ARR3⁺ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16^INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in <i>MYCN</i>-transduced retinae and <i>MYCN^A</i> retinoblastomas in patients. These findings demonstrate the <i>MYCN^A</i> retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.