Single-Cell Rna-Seq Profiling Of The Transcriptional Response To Rb Loss In The Retinoblastoma Cell Of Origin

Retinoblastoma is a rare childhood tumor initiated by biallelic inactivation of the RB1 gene and loss of functional retinoblastoma (Rb) protein. The tumors are thought to originate in cone photoreceptor precursors since Rb knockdown induces cone precursor proliferation and elicits cone-precursor-derived retinoblastoma-like tumors in orthotopic grafts. The identification of the retinoblastoma cell of origin and the authentic initiating mutation presents an opportunity to define the detailed molecular mechanisms controlling malignant transformation of a human cancer. In this project, we seek to define the transcriptional and epigenetic cell state transitions that follow Rb depletion as post-mitotic human cone precursors are induced to re-enter the cell cycle. Prospectively isolated cone precursors were transduced with lentivirus carrying either of three RB1 shRNAs or a SCR control shRNA and then re-isolated by FACS. Single cell RNA-sequencing (scRNA-seq) was performed at multiple time points over 15 days by generating full-length cDNAs corresponding to polyadenylated RNAs followed by sequencing. Among the 572 individual cells that passed quality control, an average of 6.6 million mapped reads and ~21,000 genes (~93,000 transcript isoforms) were detected per transcriptome. In addition, 300 cells per shRNA group were evaluated for genome-wide open chromatin using uATAC-seq at day 0 and 15. Cell transcriptomes were pseudotemporally ordered using “Waterfall” in order to define cellular trajectories and corresponding transcriptomic responses to Rb loss. Using transcriptomic, epigenetic, and network analyses, we define cell state transitions and identify potential mediators of such transitions, that are associated with the initiation of retinoblastoma development. Citation Format: Sunhye Lee, Hardeep P. Singh, Martin Triska, Kevin T. Stachelek, Maxwell M. Bay, Brendan H. Grubbs, Matthew E. Thronton, Haoze Hu, Neil I. Segil, Michael A. Bonaguidi, David Cobrinik. Single-cell RNA-seq profiling of the transcriptional response to Rb loss in the retinoblastoma cell of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2068.