Abstract
Purpose :
Most retinoblastomas are thought to develop from maturing cone precursors in response to biallelic RB1 loss. However, ~1.4% of retinoblastomas lack RB1 mutations but show MYCN amplification (MYCNA). MYCNA retinoblastomas are more aggressive and have distinct morphology from RB1-mutant tumors. This study aims to i) define retinal cell-type-specific responses to ectopic MYCN in explanted retinae, and ii) determine if MYCN overexpression induces behavior similar to MYCNA retinoblastoma.
Methods :
Human developing retinae were transduced with lentiviruses expressing MYCN and GFP. Retinae were EdU treated for 4 hours before harvest on Days 7-57. Ki67 and EdU were used to mark cell cycle entry. Immunofluorescence was used to evaluate cell types and cell cycle related proteins. MYCN-transduced retinal cells were orthotopically engrafted to the subretinal space in NSG mice.
Results :
Lentiviral transduction elicited MYCN expression in subset of the GFP expressing cells. ~98% of the cells that over-expressed MYCN co-stained with RXRγ and were positioned in the outer nuclear layer at early time points indicative of cone cell identity. RB protein levels did not change due to ectopic MYCN expression. Ki67 was detected in 17-78% and EdU in 8-37% of RXRγ+ cells over a time course of 10-57 days post MYCN expression. At 14 days post MYCN transduction the GFP+/Ki67+ and GFP+/EdU+ cells were predominantly RXRγ+/CRX+ and rarely NRL+, BRN3+, or PROX1+. However, similar proportions of PROX1+ cells were Ki67+ in vector-transduced retinae suggesting that these were retinal progenitor cells. No AP2a+, Syntaxin+, or PKCa+ cells were found in cell cycle. MYCN overexpression caused upregulation of cell cycle related proteins such as E2F3 in the RXRγ+ cells. By 21 days post MYCN transduction masses of proliferating RXRγ+ cells were observed and cultured to make cell lines. Orthotopic xenograft of MYCN transduced retina cells resulted in tumors in 3 of 4 mice, within 1.5 months.
Conclusions :
MYCN overexpression induces cone precursor proliferation in explanted retinae and tumorigenesis in orthotopic grafts. Tumors from MYCN transduced retinal cells appear earlier than those with RB-depletion, coinciding with the early age of diagnosis in MYCN-derived retinoblastomas in patients. This study suggests that MYCNA as well as RB1 mutant retinoblastomas derive from a cone precursor cell of origin.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.