Post-transcriptional silencing of p14ARF drives retinoblastoma malignant conversion in CRISPR-engineered retinal organoids

Purpose : This study aims to elucidate the molecular mechanisms driving malignant transformation from pre-malignant lesions, leveraging a CRISPR-engineered human retinoblastoma retinal organoid (RBRO) model that recapitulates the cone cell-of-origin and timing of multi-step retinoblastoma genesis. Background : Most retinoblastomas arise from maturing cone photoreceptor precursors (CPs) following biallelic RB1 inactivation. The process can be recapitulated in explanted fetal retina, where pRB-depleted CPs proliferate, followed by a 3-5 month indolence phase and emergence of retinoblastoma-like masses at tissue ages mirroring in vivo disease. CRISPR engineered retinal organoids (ROs) provide a promising model with which to define mechanisms that underlie malignant progression. Methods : We generated RB1 knockout iPSC lines through CRISPR editing of the GNAT2-EGFP cone reporter (Bai et al ., PMID 37902188). Chimeric RBROs were produced by mixing RB1 knockout and unedited parental iPSCs. Proliferation dynamics, cell identities and cell state changes of EGFP+ RB1 -/- cones were evaluated by live imaging of hydrogel-embedded RBROs, immunofluorescent (IF) staining, and deep full-length scRNA-seq. Additional CRISPR edits were introduced to test candidate drivers of indolence entry and escape. Results : In RB1 WT ROs, EGFP specifically, robustly and innocuously labeled post-mitotic cones. In RBROs, live imaging and IF staining revealed initial EGFP+ RB1 -/- cone proliferation followed by a pre-malignant indolence phase starting at ~d120. Most of the initially proliferating cones become Ki67-negative, with some adopting mature cone morphology. Retinoblastoma-like foci composed of cells co-expressing EGFP, cone markers, and Ki67 formed after ~d280, a tissue age that equates to the first post-natal month when retinoblastomas emerge. Single-cell transcriptomic profiling at different tumorigenesis stages revealed distinct RB1 -/- cell proliferation, cell differentiation, and cell stress states with high expression of CDKN2A ARF RNA and p14ARF protein. In contrast to initially proliferating pre-indolence CPs, the later proliferating post-indolence retinoblastoma-like cells expressed CDKN2A ARF RNA but not p14ARF protein, consistent with post-transcriptional silencing of p14ARF as a mechanism underlying indolence escape. RBROs lacking p14ARF exhibited sustained initial cone proliferation similar to indolence-escaped p14ARF-WT RBROs. Conclusion : We established a human retinoblastoma organoid model that recapitulates the cell-of-origin, developmental context, and temporal sequence of multi-step retinoblastoma genesis. This system uncovers stage-specific molecular signatures and highlights p53 pathway regulation - particularly p14ARF post-transcriptional silencing - as a key driver of the indolence-malignancy transition.