Abstract 3944: Deciphering retinoblastoma indolence-malignancy transition with <i>RB1</i> knockout cone reporter retinal organoids

Abstract Purpose: This study aims to elucidate the molecular mechanisms driving the transition from pre-malignant lesions to malignancy using a human retinoblastoma retinal organoid (RBRO) model that faithfully recapitulates the cell-of-origin and multi-step retinoblastoma genesis. Background: Retinoblastoma originates from maturing cone photoreceptor precursors with biallelic RB1 inactivation. In explanted fetal retinae, pRB-depleted post-mitotic cone precursors proliferate, followed by a 3-5 month premalignant indolence phase before retinoblastoma-like masses emerge at tissue ages mirroring in vivo disease. Research on this transition is limited by fetal tissue availability, but CRISPR engineered retinal organoids (ROs) provide a promising alternative. This study introduces a robust organoid platform to model and investigate the retinoblastoma indolence-malignancy transition. Methods: We generated cone-reporter iPSC lines through CRISPR knock-in of EGFP-P2A at the GNAT2 locus. A second round of CRISPR editing produced homozygous RB1 knockout. Chimeric RB1+/+ ROs and RB1-/- RBROs were generated from edited iPSCs mixed with unedited parental iPSCs. ROs and RBROs were embedded in hydrogel and live-imaged episodically to track EGFP+ cone proliferation dynamics. Deep full-length scRNA-seq was carried out on FACS isolated EGFP+ RB1+/+ and RB1-/- cones at key developmental and tumorigenesis stages, with results validated by immunofluorescent staining. Results: In RB1 WT ROs, EGFP specifically, robustly and innocuously labeled immature and mature cones. In RBROs, bi-weekly live confocal imaging revealed initial EGFP+ RB1-/- cone proliferation followed by a pre-malignant indolence phase starting at ∼d150. The majority of the initially proliferating cones were Ki67-negative with some adopting mature cone morphology. Nascent retinoblastoma-like foci co-expressing EGFP, cone markers, and Ki67 formed after ∼d280, a tissue age that equates to the first post-natal month when early retinoblastomas typically emerge. Single cell transcriptomics of RB1-/- cones from multiple tumorigenesis stages showed distinct molecular signatures of proliferation, differentiation and stress cell states and suggest a role of p53 pathway in indolence entry and escape. Immunofluorescent staining suggested post-transcriptional silencing of p14ARF underlying indolence escape. Conclusion: We established a human retinoblastoma organoid model that recapitulates the cell-of-origin and timing of multi-step retinoblastomagenesis. This model revealed distinct molecular signatures at each tumorigenesis stage and highlighted altered expression of a p53 pathway regulator during the retinoblastoma indolence-malignancy transition. Citation Format: Jinlun Bai, David S. Koos, Kayla Stepanian, Kevin Stachelek, Bhavana Bhat, Scott E. Fraser, Rex A. Moats, David Cobrinik. Deciphering retinoblastoma indolence-malignancy transition with RB1 knockout cone reporter retinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3944.