Distinct rod and cone precursor transcriptomic responses to pRB loss reveals a metabolic predisposition to retinoblastoma initiation

Abstract

Purpose : Cone precursors (CPs) form retinoblastomas in response to RB1 loss, while rod precursors (RPs) do not. Transcriptomic features that distinguish CP versus RP responses are poorly defined. Here we examined time-dependent gene expression changes of pRB-depleted fetal CP and RPs to identify transcriptomic changes that control retinoblastoma initiation.

Methods : Human fetal retina photoreceptor precursors were FACS isolated, transduced with shRNAs targeting RB1 (shRB1) or a non-targeting control (shSCR), and subjected to SMART-Seq full-length scRNAseq at 4 - 12 days post-transduction. Principal components that segregate transcriptomes according to culture time and pRB knockdown status were identified and cell states defined by hierarchical clustering, pseudotemporal trajectory reconstruction, and differential gene expression analysis. Explanted pRB-depleted retina was treated with PGMI-004A followed by immunofluorescent analysis of Ki67, Cyclin A, and Cyclin B expression and EdU incorporation.

Results : Pseudotemporal trajectories were deduced and gene expression changes identified for pRB-depleted CPs and RPs. Among other changes, we found a 3-fold reduction of PGAM1 RNA in RPs versus a 1.2-fold reduction in CPs. As PGAM1 regulates glycolysis and nucleotide synthesis, we determined if PGAM activity is needed for CP proliferation. Compared to retinae transduced with shRB1 alone, those treated with PGAM1 inhibitor PGMI-004A had an increased percentage of Ki67+ CPs (43.7% vs 27.9%) but no EdU incorporation (0.0% vs 24.8%, p < 0.001, Fisher's exact test for both). PGMI-004A-treated pRB-depleted Ki67+ cone precursors had more frequent co-expression of S phase marker Cyclin A2 (73.7% vs 59.2%) and reduced co-expression of G2/M marker Cyclin B (20.4% vs 62.8%, p < 0.05, Fisher’s exact test for both).

Conclusions : CP and RP transcriptomic responses to RB1 loss were established. PGAM1 expression was abrogated in pRB-depleted RPs but persisted in pRB-depleted CPs. PGAM1 activity was not required for CP cell cycle entry but was needed for nucleotide incorporation and progression past S phase, These findings demonstrate that cell type-specific responses to pRB loss underlie the cone precursor’s tumorigenic potential and suggest that cone-specific regulation of metabolic proteins is critical in retinoblastoma initiation.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.