Project Summary Understanding how a normal cell becomes a malignant cancer may provide new therapeutic and prevention opportunities. However, although many genetic changes that promote cancer have been identified, and the corresponding signaling pathways elucidated in model settings, the cellular responses that mediate oncogenic transformation in an authentic human cancer cell-of-origin are largely unexplored. This study aims to define cellular responses that mediate the development of a childhood eye cancer, retinoblastoma, which is well suited to such analyses because the cell-of-origin (the maturing cone precursor), the major initiating mutation (biallelic RB1 inactivation), and an indolent pre-malignant stage have been identified, and because the cell-of- origin’s proliferative response to initiating oncogenic mutations, progression through a pre-malignant state, and emergence as a retinoblastoma tumor can be observed and characterized in in vitro and in vivo assays. The proposed studies will explore the successive gene expression and cell signaling changes that underlie the cone precursors’ responses to RB1 loss, including changes that mediate cell cycle entry, proliferation, retreat to an indolent pre-malignant state, and either deactivation to form a senescence-like retinoma or reactivation to form a highly proliferating retinoblastoma mass. Aim 1 will define the transcriptomic changes that occur in response to RB loss as the cone precursor cell of origin embarks on its malignant trajectory in the developing retina. Aims 2 and 3 will focus on recently identified early gene expression responses and their importance for cone precursor proliferation and traversal of the pre-malignant phase. Aim 2 centers on the up-regulation of an oncogenic epigenetic regulator, which may enable reactivation of indolent, pre-malignant cone precursors and emergence of retinoblastoma tumors. Aim 3 centers on the down-regulation of a negative regulator of PI3- kinase signaling, which may enhance the RB-deficient cone precursor survival and transformation. Aim 4 will focus on the RB structures, biochemical functions, and downstream mechanisms that are needed to suppress cone precursor cell cycle entry and traversal of the pre-malignant phase. Particular attention will be paid to the stages at which different RB functions contribute to suppress retinoblastoma and the gene expression and cell signaling changes that mediate RB effects. Together, the proposed studies are expected to improve understanding of the oncogenic responses of a human cancer cell-of-origin during its journey towards malignancy. In turn, the improved understanding may reveal vulnerabilities that can be targeted in order to prevent retinoblastoma in genetically predisposed children.