Post-Transcriptional Silencing of p14ARF Drives Retinoblastoma Malignant Conversion

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 CDKN2AARF RNA and p14ARF protein. In contrast to initially proliferating pre-indolence CPs, the later proliferating post-indolence retinoblastoma-like cells expressed CDKN2AARF 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. Citation Format: Jinlun Bai, David S. Koos, Kevin Stachelek, Bhavana Bhat, Susan Asatrian, Patrick Belen, Sunjum Sanghari, Kayla Stepanian, Scott Fraser, Rex A. Moats, David Cobrinik, . Post-transcriptional silencing of p14ARF drives retinoblastoma malignant conversion in CRISPR-engineered retinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6182.