Retinoblastoma is the most common pediatric eye cancer and is caused by the biallelic inactivation of the tumor suppressor gene RB1. There are between 200 and 300 new patients with retinoblastoma in the U.S. each year, most diagnosed before two years of age. When caught early, and with proper care, survival from retinoblastoma is relatively high. However, treatments, including enucleations, or removal of the eye, have lasting effects on the lives of the children. Furthermore, retinoblastomas with extraocular involvement have a much poorer prognosis. While animal models of retinoblastoma have been useful for preclinical and clinical testing of retinoblastoma treatment, mice with loss of RB1 never develop retinoblastoma and therefore cannot accurately recapitulate human retinoblastoma formation. This has led to gaps in our understanding of the tumor initiation, development, and cellular specificity of human retinoblastoma. The goal of this study is to use our new model of spontaneous human retinoblastoma using patient derived iPSCs to study the developmental origin(s) of retinoblastoma and characterize the mechanisms of the second hit mutation. We have generated 15 induced pluripotent stem cell (iPSC) lines derived from patients with germline mutations in RB1. Using an optimized retinal differentiation culture, we have differentiated the patient lines into retina, dissociated the organoids, injected the cells into mice to screen for tumor formation. The resulting tumors have expression patterns similar to retinoblastoma patient derived xenografts. I now have a model system to study retinoblastoma tumor formation during retinal differentiation. I will use our new retinoblastoma reporter line in H9 hESCs, SYK-GFP, and a combination of immunostaining, single cell sequencing and electron microscopy to determine cell of origin or retinoblastoma. Additionally, I will analyze the tumors over multiple rounds of differentiation to determine the mechanism of the second hit and if there is any genetic predisposition to the loss of heterozygosity that occurs in patients with germline RB1 mutations. This study will allow us, for the first time, to answer question about retinoblastoma initiation and progression. Defining the cell of origin or retinoblastoma and the mechanism of the second hit of RB1 will not only increase our depth of understanding of retinoblastoma development but can be more generally applied to the fundamental understanding of cancer initiation. Furthermore, the data provided by this study will hopefully provide insight to create more targeted therapies for children with retinoblastoma.
Retinoblastoma, the most common pediatric eye tumor, has been difficult to study based on a lack of an accurate model of retinoblastoma initiation. Therefore, I have created a new model for studying spontaneous human retinoblastoma development using patient derived induced pluripotent stem cells in a retinal organoid culture. Using this system I will study the natural development of the disease and determine the cell of origin and the genetic mechanisms of oncogenic transformation of retinoblastoma.