Abstract 1726: Structure-function analysis of retinoblastoma tumor suppressor protein in human cone precursors

Abstract Background: The retinoblastoma protein, pRB, is a key cell cycle governor that is functionally impaired in most cancers, but its mechanism of suppressing tumorigenesis in a tumor cell-of-origin has not been characterized. This study aims to define pRB structural features and corresponding cellular functions that suppress cone precursor (CP) proliferation, likely the first step in retinoblastomagenesis. Method: Intact human fetal retinae were co-transduced with lentiviruses to knockdown endogenous pRB and ectopically express wild type or structure-based substitution mutant pRBs. The ectopically expressed pRB mutants had selectively impaired interactions with E2F transactivation domains (ΔG), with the E2F1 “marked box” domain (ΔS), or with the LxCxE protein interaction motif (ΔL). At day 12-75 post-infection, transduced retinae were analyzed by quantitative immunofluorescence staining of Ki67/EdU, pRB and CP marker L/M-opsin. Results: In intact retina, pRB knockdown induced cell cycle re-entry of L/M-opsin+ postmitotic cone precursors, whereas those reconstituted with wild type pRB did not express Ki67 indicative of a block to cell cycle entry. Unexpectedly, all three mutants (pRB-ΔG, -ΔL, and -ΔS) were able to block cell cycle re-entry when expressed at the high-normal range the of endogenous pRB. However, at the low-normal expression level, ectopic wild type but not mutant pRBs suppressed proliferation. Conclusion: All three surface targeted mutants (pRB-ΔG, ΔL, and ΔS) retain partial ability to suppress cone precursor cell cycle entry. This may be attributed to the presence of multiple independent cell cycle inhibitory functions. These findings suggest that pRB-mediated regulation of E2F-responsive cell cycle genes, interactions with LxCxE motif-containing proteins, and EZH2 mediated repression of repetitive elements are individually dispensable for suppressing aberrant cone precursor proliferation. Citation Format: Sijia Wang, Brendan Grubbs, Matthew Thornton, David Cobrinik. Structure-function analysis of retinoblastoma tumor suppressor protein in human cone precursors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1726.